Tailored Oils

ABSTRACT

Recombinant DNA techniques are used to produce oleaginous recombinant cells that produce triglyceride oils having desired fatty acid profiles and regiospecific or stereospecific profiles. Genes manipulated include those encoding stearoyl-ACP desturase, delta 12 fatty acid desaturase, acyl-ACP thioesterase, ketoacyl-ACP synthase, and lysophosphatidic acid acyltransferase. The oil produced can have enhanced oxidative or thermal stability, can be useful as a frying oil, shortening, roll-in shortening, tempering fat, cocoa butter replacement, as a lubricant, or as a feedstock for various chemical processes. The fatty acid profile can be enriched in midchain profiles or the oil can be enriched in triglycerides of the saturated-unsaturated-saturated type.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 61/635,285, filed Apr. 18, 2012, U.S.Provisional Patent Application No. 61/639,838, filed Apr. 27, 2012, U.S.Provisional Patent Application No. 61/655,469, filed Jun. 4, 2012, U.S.Provisional Patent Application No. 61/672,196, filed Jul. 16, 2012, U.S.Provisional Patent Application No. 61/679,026, Aug. 2, 2012, U.S.Provisional Patent Application No. 61/715,998, filed Oct. 19, 2012, U.S.Provisional Patent Application No. 61/769,678, filed Feb. 26, 2013, andU.S. Provisional Patent Application No. 61/778,963, filed Mar. 13, 2013,all of which are incorporated by reference herein with the proviso thatthe definitions of terms herein shall be the complete and controllingdefinitions.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as shown in pages 1-24,appended hereto.

FIELD OF THE INVENTION

Embodiments of the present invention relate to oils/fats, fuels, foods,and oleochemicals and their production from cultures of geneticallyengineered cells. Specific embodiments relate to oils with a highcontent of triglycerides bearing fatty acyl groups upon the glycerolbackbone in particular regiospecific patterns, highly stable oils, oilswith high levels of mid-chain fatty acids, and products produced fromsuch oils.

BACKGROUND OF THE INVENTION

PCT Publications WO2008/151149, WO2010/06032, WO2011/150410,WO2011/150411, and international patent application PCT/US12/23696disclose oils and methods for producing those oils in microbes,including microalgae. These publications also describe the use of suchoils to make oleochemicals and fuels.

Tempering is a process of converting a fat into a desired polymorphicform by manipulation of the temperature of the fat or fat-containingsubstance, most commonly used in chocolate making.

Certain enzymes of the fatty acyl-CoA elongation pathway function toextend the length of fatty acyl-CoA molecules. Elongase complex enzymesextend fatty acyl-CoA molecules in 2 carbon additions, for examplemyristoyl-CoA to palmitoyl-CoA, stearoyl-CoA to arachidyl-CoA, oroleyl-CoA to eicosanoyl-CoA, eicosanoyl-CoA to erucyl-CoA. In addition,elongase enzymes also extend acyl chain length in 2 carbon increments.KCS enzymes condense acyl-CoA molecules with two carbons frommalonyl-CoA to form beta-ketoacyl-CoA. KCS and elongases may showspecificity for condensing acyl substrates of particular carbon length,modification (such as hydroxylation), or degree of saturation. Forexample, the jojoba (Simmondsia chinensis) beta-ketoacyl-CoA synthasehas been demonstrated to prefer monounsaturated and saturated C18- andC20-CoA substrates to elevate production of erucic acid in transgenicplants (Lassner et al., Plant Cell, 1996, Vol 8(2), pp 281-292), whereasspecific elongase enzymes of Trypanosoma brucei show preference forelongating short and midchain saturated CoA substrates (Lee et al.,Cell, 2006, Vol 126(4), pp 691-9).

The type II fatty acid biosynthetic pathway employs a series ofreactions catalyzed by soluble proteins with intermediates shuttledbetween enzymes as thioesters of acyl carrier protein (ACP). Bycontrast, the type I fatty acid biosynthetic pathway uses a single,large multifunctional polypeptide.

The oleaginous, non-photosynthetic alga, Protetheca moriformis, storescopious amounts of triacylglyceride oil under conditions when thenutritional carbon supply is in excess, but cell division is inhibiteddue to limitation of other essential nutrients. Bulk biosynthesis offatty acids with carbon chain lengths up to C18 occurs in the plastids;fatty acids are then exported to the endoplasmic reticulum whereelongation past C18 and incorporation into triacylglycerides (TAGs) isbelieved to occur. Lipids are stored in large cytoplasmic organellescalled lipid bodies until environmental conditions change to favorgrowth, whereupon they are rapidly mobilized to provide energy andcarbon molecules for anabolic metabolism.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a natural oil produced inan optionally plastidic oleaginous microbe, the oil having 3%, 2%, 1%,0.5%, or 0.3% or less of polyunsaturated fatty acids. In some cases, theoil is stable at 110° C. so that the inflection point in conductance isnot yet reached by 20 hours under conditions of the AOCS Cd 12b-92Rancimat test. In some cases, the oil is stable at 110° C. so that theinflection point in conductance is not yet reached by 5 days underconditions of the AOCS Cd 12b-92 Rancimat test, when 1050 ppm oftocopherol and 500 pm of ascorbyl palmitate are added to the oil. Insome cases, the oil is produced by cultivating cells having a reducedfatty acid desaturase activity so as to reduce the production oflinoleic acid by the cell. In some cases, the cell is auxotrophic orpartially auxotrophic for linoleic acid. In some cases, linoleic acidsynthesis is under regulatable control.

In another aspect, the present invention provides a plastidic oleaginousmicrobial cell capable of production of at least 20% lipid by dry cellweight when cultivated heterotrophically, and wherein the cell comprisesrecombinant nucleic acids operable to suppress the activity of a fattyacid desaturase gene product so that the cell produces an oil with atriacylglycerol profile having less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%,or 0.2% linoleic acid. In some cases, the cell is a linoleic acidauxotroph or linoleic acid synthesis can be regulated via environmentalconditions.

In another aspect, the present invention provides an oil produced in aplastid of the cells discussed above. In some cases, the oil is stableat 110° C. so that the inflection point in conductance is not yetreached by 20 hours under conditions of the AOCS Cd 12b-92 Rancimattest.

In another aspect, the present invention provides a method forincreasing the proportion of long chain fatty acids in triglycerides ofa cell oil, in which the method comprises reducing the activity of anendogenous acyl-ACP thioesterase having a preference for fatty-ACPchains of length C18. In some cases, reducing the activity of anendogenous acyl-ACP thioesterase comprises a knockout or knockdown ofthe acyl-ACP thioesterase. In some cases, the knockdown uses a hairpinRNA. In some cases, the method further comprises providing a cellcomprising recombinant nucleic acids operable to reduce stearoyl-ACPdesaturase activity in the cell. In some cases, the stearate levels inthe cell are increased due to the recombinant nucleic acids. In somecases, the cell produces a storage oil with a triglyceride fatty acidprofile of at least 20, 30, 40, 50, 60, 70, 80, or 90% stearate. In somecases, the storage oil is at least 20, 30, 40, 50, 60, 70, 80, or 90%SOS.

In another aspect, the present invention provides a plastidic oleaginousmicrobial cell culture of cells comprising recombinant nucleic acidsoperable to cause the cells to produce a triglyceride rich oil, whereinthe triglycerides of the oil have a laurate content in excess of 40, 50,60, 70, 80, or 90%. In some cases, the recombinant nucleic acidscomprise a laurate preferring acyl-ACP thioesterase and a LPAAT operableto add laurate to the sn-2 position so as to form trilaurin. In somecases, the recombinant nucleic acids are operable to reduce theexpression of one or more of an endogenous acyl-ACP thioesterase and anendogenous LPAAT.

In another aspect, the present invention provides a natural fat producedfrom recombinant cultivated cells, whereinSaturated-Unsaturated-Saturated triglycerides such as SOS, POP, and/orPOS make up at least 50% of the fat. In some cases, the fat forms βpolymorph and/or β′ polymorph crystals. In some cases, the fat has a 2Lor 3L lamellae structure. In some cases, the triglycerides of the fathave a fatty acid profile characterized in that the sum of thepercentage of stearate and palmitate is equal to the percentage ofoleate multiplied by 2.0+/−40%. In some cases, the fat has greater than65% SOS, less than 45% unsaturated fatty acid, less than 5% unsaturatedfatty acids, less than 1% lauric acid, and less than 2% trans fattyacid. In some cases, the sum of the percent stearate and palmitate inthe fatty acid profile of the fat is twice the percentage of oleate,±20%. In some cases, the sn-2 profile of the fat is at least 40% oleate.In some cases, the fat is at least 40, 50, 60, 70, 80, or 90% SOS.

In another aspect, the present invention provides a method comprisingusing any one of the fats discussed above as a cocoa butter replacer,substitute or extender in a confectionary product.

In another aspect, the present invention provides a natural oil producedby a plastidic oleaginous microbe comprising recombinant nucleic acidsoperable to express an exogenous LPAAT so as to alter the amount of oiland/or the fatty acid profile of the oil, wherein the microbe optionallycomprises exogenous nucleic acids operable to express an exogenous KASIIor to decrease the activity one or more fatty acid desaturase.

In another aspect, the present invention provides a natural oil producedby a plastidic oleaginous microbial cell comprising recombinant nucleicacids operable to express an exogenous elongase so as to alter the fattyacid profile, TAG profile, stereospecific profile, or regiospecificprofile of triglycerides produced by the cell.

In another aspect, the present invention provides a method for producingan oil or fat, in which the method comprises: (a) cultivating arecombinant oleaginous cell in a growth phase under a first set ofconditions that is permissive to cell division so as to increase thenumber of cells due to the presence of a fatty acid; (b) cultivating thecell in an oil production phase under a second set of conditions that isrestrictive to cell division but permissive to production of an oil thatis depleted in the fatty acid; and (c) extracting the oil from the cell,wherein the cell has a mutation or exogenous nucleic acids operable tosuppress the activity of a fatty acid synthesis enzyme, the enzymeoptionally being a stearoyl-ACP desaturase, delta 12 fatty aciddesaturase, ketoacyl-ACP synthase, or LPAAT, the fatty acid optionallybeing oleic, linoleic, linolenic, stearic, or palmitic. In some cases,the fatty acid is depleted in the oil by at least than 50, 60, 70, 80,or 90%. In some cases, the cell is cultivated heterotrophically. In somecases, the cell is a microalgal cell. In some cases, the cell producesat least 40, 50, 60, 70, 80, or 90% oil by dry cell weight.

In another aspect, the present invention provides an oil produced by theabove method.

In another aspect, the present invention provides a food, chemical orfuel produced from the above oil.

These and other aspects and embodiments of the invention are describedand/or exemplified in the accompanying drawings, a brief description ofwhich immediately follows, the detailed description of the invention,and in the examples. Any or all of the features discussed above andthroughout the application can be combined in various embodiments of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-14 show fatty acid profiles and melting curves of refined,bleached and deodorized oils from genetically engineered Protothecamoriformis strains, as discussed in Example 4;

FIG. 15 shows the stability of different oils as a function ofantioxidant concentration, as discussed in Example 5;

FIG. 16 shows various properties of natural oils with very low levels ofpolyunsaturated fatty acids in accordance with an embodiment of theinvention; and

FIG. 17 shows a plot of percent solid fat content for various oils asfollows: (a) P. moriformis RBD oil without lipid pathway engineering;(b) Brazilian cocoa butter+25% milkfat; (c) three replicates of P.moriformis RBD oil from a strain expressing hairpin nucleic acids thatreduce levels of a SAD allele thus reducing oleic acid and increasingstearic acid in the TAG profile; (d) P. moriformis RBD oil from a strainoverexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, seeExample 45); (e) Malaysian cocoa butter+25% milkfat; and (f) Malaysiancocoa butter. The cocoa butter and cocoa butter milkfat values areliterature values (Bailey's Industrial Oils and Fat Products, 6^(th)ed.).

FIG. 18 shows the results of thermal stability testing performed onmethylated oil prepared from high-oleic (HO) and high-stabilityhigh-oleic (HSAO) triglyceride oils prepared from heterotrophicallygrown oleaginous microalgae, in comparison to a soya methyl estercontrol sample.

FIG. 19 shows various properties of high-oleic and high-stabilityhigh-oleic algal oils.

FIG. 20 shows TAG composition of S4495, S5665 and S5675 oils from flaskand fermentor biomass. La=laurate (C12:0), M=myristate (C14:0),P=palmitate (C16:0), Po=palmitoleate (C16:1), S=stearate (C18:0),O=oleate (C18:1), L=linoleate (C18:2), Ln=α-linolenate (C18:3),A=arachidate (C20:0), B=behenate (C22:0), Lg=lignocerate (C24:0),Hx=hexacosanoate (C26:0) S-S-S refers to the sum of TAGs in which allthree fatty acids are saturated. In each block of bars, the strains areshown in the order illustrated at the bottom of the figure.

FIG. 21 shows TAG composition of S5774, S5775 and S5776 oils from shakeflask biomass. La=laurate (C12:0), M=myristate (C14:0), P=palmitate(C16:0), Po=palmitoleate (C16:1), S=stearate (C18:0), O=oleate (C18:1),L=linoleate (C18:2), Ln=α-linolenate (C18:3), A=arachidate (C20:0),B=behenate (C22:0), Lg=lignocerate (C24:0), Hx=hexacosanoate (C26:0).S-S-S refers to the sum of TAGs in which all three fatty acids aresaturated. In each block of bars, the strains are shown in the orderillustrated at the bottom of the figure.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

An “allele” is any of one or more alternative forms of a gene whichrelate to one trait or characteristic.

A “natural oil” or “natural fat” shall mean a predominantly triglycerideoil obtained from an organism, where the oil has not undergone blendingwith another natural or synthetic oil, or fractionation so as tosubstantially alter the fatty acid profile of the triglyceride. Inconnection with an oil comprising triglycerides of a particularregiospecificity, the natural oil or natural fat has not been subjectedto interesterification or other synthetic process to obtain thatregiospecific triglyceride profile, rather the regiospecificity isproduced naturally, by a cell or population of cells. In connection witha natural oil or natural fat, and as used generally throughout thepresent disclosure, the terms oil and fat are used interchangeably,except where otherwise noted. Thus, an “oil” or a “fat” can be liquid,solid, or partially solid at room temperature, depending on the makeupof the substance and other conditions. Here, the term “fractionation”means removing material from the oil in a way that changes its fattyacid profile relative to the profile produced by the organism, howeveraccomplished. The terms “natural oil” and “natural fat” encompass suchoils obtained from an organism, where the oil has undergone minimalprocessing, including refining, bleaching and/or degumming, that doesnot substantially change its triglyceride profile. A natural oil canalso be a “noninteresterified natural oil”, which means that the naturaloil has not undergone a process in which fatty acids have beenredistributed in their acyl linkages to glycerol and remain essentiallyin the same configuration as when recovered from the organism.

“Exogenous gene” shall mean a nucleic acid that codes for the expressionof an RNA and/or protein that has been introduced into a cell (e.g. bytransformation/transfection), and is also referred to as a “transgene”.A cell comprising an exogenous gene may be referred to as a recombinantcell, into which additional exogenous gene(s) may be introduced. Theexogenous gene may be from a different species (and so heterologous), orfrom the same species (and so homologous), relative to the cell beingtransformed. Thus, an exogenous gene can include a homologous gene thatoccupies a different location in the genome of the cell or is underdifferent control, relative to the endogenous copy of the gene. Anexogenous gene may be present in more than one copy in the cell. Anexogenous gene may be maintained in a cell as an insertion into thegenome (nuclear or plastid) or as an episomal molecule.

“Fatty acids” shall mean free fatty acids, fatty acid salts, or fattyacyl moieties in a glycerolipid. It will be understood that fatty acylgroups of glycerolipids can be described in terms of the carboxylic acidor anion of a carboxylic acid that is produced when the triglyceride ishydrolyzed or saponified.

“Fixed carbon source” is a molecule(s) containing carbon, typically anorganic molecule that is present at ambient temperature and pressure insolid or liquid form in a culture media that can be utilized by amicroorganism cultured therein. Accordingly, carbon dioxide is not afixed carbon source.

“In operable linkage” is a functional linkage between two nucleic acidsequences, such a control sequence (typically a promoter) and the linkedsequence (typically a sequence that encodes a protein, also called acoding sequence). A promoter is in operable linkage with an exogenousgene if it can mediate transcription of the gene.

“Microalgae” are microbial organisms that contain a chloroplast or otherplastid, and optionally that is capable of performing photosynthesis, ora prokaryotic microbial organism capable of performing photosynthesis.Microalgae include obligate photoautotrophs, which cannot metabolize afixed carbon source as energy, as well as heterotrophs, which can livesolely off of a fixed carbon source. Microalgae include unicellularorganisms that separate from sister cells shortly after cell division,such as Chlamydomonas, as well as microbes such as, for example, Volvox,which is a simple multicellular photosynthetic microbe of two distinctcell types. Microalgae include cells such as Chlorella, Dunaliella, andPrototheca. Microalgae also include other microbial photosyntheticorganisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena,and Pyrobotrys. Microalgae also include obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate algae species and species of the genusPrototheca.

In connection with a recombinant cell, the term knockdown refers to agene that has been partially suppressed (e.g., by about 1-95%) in termsof the production or activity of a protein encoded by the gene.

Also, in connection with a recombinant cell, the term knockout refers toa gene that has been completely or nearly completely (e.g., >95%)suppressed in terms of the production or activity of a protein encodedby the gene. Knockouts can be prepared by homologous recombination of anoncoding sequence into a coding sequence, gene deletion, mutation orother method.

An “oleaginous” cell is a cell capable of producing at least 20% lipidby dry cell weight, naturally or through recombinant or classical strainimprovement. An “oleaginous microbe” or “oleaginous microorganism” is amicrobe, including a microalga that is oleaginous.

An “ordered oil” or “ordered fat” is one that forms crystals that areprimarily of a given polymorphic structure. For example, an ordered oilor ordered fat can have crystals that are greater than 50%, 60%, 70%,80%, or 90% of the β or β′ polymorphic form.

In connection with a natural oil, a “profile” is the distribution ofparticular species or triglycerides or fatty acyl groups within the oil.A “fatty acid profile” is the distribution of fatty acyl groups in thetriglycerides of the oil without reference to attachment to a glycerolbackbone. Fatty acid profiles are typically determined by conversion toa fatty acid methyl ester (FAME), followed by gas chromatography (GC)analysis with flame ionization detection (FID). The fatty acid profilecan be expressed as one or more percent of a fatty acid in the totalfatty acid signal determined from the area under the curve for thatfatty acid. FAME-GC-FID measurement approximate weight percentages ofthe fatty acids. A “sn-2 profile” is the distribution of fatty acidsfound at the sn-2 position of the triacylglycerides in the oil. A“regiospecific profile” is the distribution of triglycerides withreference to the positioning of acyl group attachment to the glycerolbackbone without reference to stereospecificity. In other words, aregiospecific profile describes acyl group attachment at sn-1/3 vs.sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate)and SOP (stearate-oleate-palmitate) are treated identically. A“stereospecific profile” describes the attachment of acyl groups atsn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such asSOP and POS are to be considered equivalent. A “TAG profile” is thedistribution of fatty acids found in the triglycerides with reference toconnection to the glycerol backbone, but without reference to theregiospecific nature of the connections. Thus, in a TAG profile, thepercent of SSO in the oil is the sum of SSO and SOS, while in aregiospecific profile, the percent of SSO is calculated withoutinclusion of SOS species in the oil. In contrast to the weightpercentages of the FAME-GC-FID analysis, triglyceride percentages aretypically given as mole percentages; that is the percent of a given TAGmolecule in a TAG mixture.

“Recombinant” is a cell, nucleic acid, protein or vector that has beenmodified due to the introduction of an exogenous nucleic acid or thealteration of a native nucleic acid. Thus, e.g., recombinant cells canexpress genes that are not found within the native (non-recombinant)form of the cell or express native genes differently than those genesare expressed by a non-recombinant cell. Recombinant cells can, withoutlimitation, include recombinant nucleic acids that encode for a geneproduct or for suppression elements such as mutations, knockouts,antisense, interfering RNA (RNAi) or dsRNA that reduce the levels ofactive gene product in a cell. A “recombinant nucleic acid” is a nucleicacid originally formed in vitro, in general, by the manipulation ofnucleic acid, e.g., using polymerases, ligases, exonucleases, andendonucleases, using chemical synthesis, or otherwise is in a form notnormally found in nature. Recombinant nucleic acids may be produced, forexample, to place two or more nucleic acids in operable linkage. Thus,an isolated nucleic acid or an expression vector formed in vitro byligating DNA molecules that are not normally joined in nature, are bothconsidered recombinant for the purposes of this invention. Once arecombinant nucleic acid is made and introduced into a host cell ororganism, it may replicate using the in vivo cellular machinery of thehost cell; however, such nucleic acids, once produced recombinantly,although subsequently replicated intracellularly, are still consideredrecombinant for purposes of this invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid.

The terms “triglyceride”, “triacylglyceride” and “TAG” are usedinterchangeably as is known in the art.

II. General

Illustrative embodiments of the present invention feature oleaginouscells that produce altered fatty acid profiles and/or alteredregiospecific distribution of fatty acids in glycerolipids, and productsproduced from the cells. Examples of oleaginous cells include microbialcells having a type II fatty acid biosynthetic pathway, includingplastidic oleaginous cells such as those of oleaginous algae. Specificexamples of cells include heterotrophic or obligate heterotophicmicroalgae of the phylum Chlorpophya, the class Trebouxiophytae, theorder Chlorellales, or the family Chlorellacae. Examples of oleaginousmicroalgae are provided in Published PCT Patent ApplicationsWO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411, includingspecies of Chlorella and Prototheca, a genus comprising obligateheterotrophs. The oleaginous cells can be, for example, capable ofproducing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cellweight, ±5%. Optionally, the oils produced can be low in DHA or EPAfatty acids. For example, the oils can comprise less than 5%, 2%, or 1%DHA and/or EPA. The above-mentioned publications also disclose methodsfor cultivating such cells and extracting oil, especially frommicroalgal cells; such methods are applicable to the cells disclosedherein and incorporated by reference for these teachings. Whenmicroalgal cells are used they can be cultivated autotrophically (unlessan obligat heterotroph) or in the dark using a sugar (e.g., glucose,fructose and/or sucrose) In any of the embodiments described herein, thecells can be heterotrophic cells comprising an exogenous invertase geneso as to allow the cells to produce oil from a sucrose feedstock.Alternately, or in addition, the cells can metabolize xylose fromcellulosic feedstocks. For example, the cells can be geneticallyengineered to express one or more xylose metabolism genes such as thoseencoding an active xylose transporter, a xylulose-5-phosphatetransporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenaseand a xylose reductase. See WO2012/154626, “GENETICALLY ENGINEEREDMICROORGANISMS THAT METABOLIZE XYLOSE”, published Nov. 15, 2012.

The oleaginous cells express one or more exogenous genes encoding fattyacid biosynthesis enzymes. As a result, some embodiments feature naturaloils that were not obtainable from a non-plant or non-seed oil, or notobtainable at all.

The oleaginous cells produce a storage oil, which is primarilytriacylglyceride and may be stored in storage vesicles of the cell. Araw oil may be obtained from the cells by disrupting the cells andisolating the oil. WO2008/151149, WO2010/06032, WO2011/150410, andWO2011/1504 disclose heterotrophic cultivation and oil isolationtechniques. For example, oil may be obtained by cultivating, drying andpressing the cells. The oils produced may be refined, bleached anddeodorized (RBD) as known in the art or as described in WO2010/120939.The raw or RBD oils may be used in a variety of food, chemical, andindustrial products or processes. After recovery of the oil, a valuableresidual biomass remains. Uses for the residual biomass include theproduction of paper, plastics, absorbents, adsorbents, as animal feed,for human nutrition, or for fertilzer.

Where a fatty acid profile of a triglyceride (also referred to as a“triacylglyceride” or “TAG”) cell oil is given here, it will beunderstood that this refers to a nonfractionated sample of the storageoil extracted from the cell analyzed under conditions in whichphospholipids have been removed or with an analysis method that issubstantially insensitive to the fatty acids of the phospholipids (e.g.using chromatography and mass spectrometry). The oil may be subjected toan RBD process to remove phospholipids, free fatty acids and odors yethave only minor or negligible changes to the fatty acid profile of thetriglycerides in the oil. Because the cells are oleaginous, in somecases the storage oil will constitute the bulk of all the TAGs in thecell. Examples 1, 2, and 8 below give analytical methods for determiningTAG fatty acid composition and regiospecific structure.

Broadly categorized, certain embodiments of the invention include (i)auxotrophs of particular fatty acids; (ii) cells that produce oilshaving low concentrations of polyunsaturated fatty acids, includingcells that are auxotrophic for unsaturated fatty acids; (iii) cellsproducing oils having high concentrations of particular fatty acids dueto expression of one or more exogenous genes encoding enzymes thattransfer fatty acids to glycerol or a glycerol ester; (iv) cellsproducing regiospecific oils, and (v) genetic constructs or cellsencoding a newly discovered gene encoding an LPAAT enzyme from CupheaPSR23 (see Example 43). The embodiments also encompass the oils made bysuch cells, the residual biomass from such cells after oil extraction,olecochemicals, fuels and food products made from the oils and methodsof cultivating the cells.

In any of the embodiments below, the cells used are optionally cellshaving a type II fatty acid biosynthetic pathway such as microalgalcells including heterotrophic or obligate heterotrophic microalgalcells, including cells classified as Chlorophyta, Trebouxiophyceae,Chlorellales, Chlorellaceae, or Chlorophyceae. In specific embodiments,the cell is of the species Prototheca moriformis, Prototheca krugani,Prototheca stagnora or Prototheca zopfii or has a 23S rRNA sequence withat least 70, 75, 80, 85 or 95% nucleotide identity to one or more of SEQID NOs: 11-19 of WO2010/063032, incorporated by reference herein as tothese sequences. By cultivating in the dark or using an obligateheterotroph, the natural oil produced can be low in chlorophyll or othercolorants. For example, the natural oil can have less than 100, 50, 10,5, 1, 0.0.5 ppm of chlorophyll without substantial purification.

The stable carbon isotope value δ13C is an expression of the ratio of¹³C/¹²C relative to a standard (e.g. PDB, carbonite of fossil skeletonof Belemnite americana from Peedee formation of South Carolina). Thestable carbon isotope value δ13C(‰) of the oils can be related to theδ13C value of the feedstock used. In some embodiments the oils arederived from oleaginous organisms heterotrophically grown on sugarderived from a C4 plant such as corn or sugarcane. In some embodimentsthe δ13C(‰) of the oil is from −10 to −17‰ or from −13 to −16‰.

In specific embodiments and examples discussed below, one or more fattyacid synthesis genes (e.g., encoding an acyl-ACP thioesterase, aketo-acyl ACP synthase, an LPAAT, a stearoyl ACP desaturase, or othersdescribed herein) is incorporated into a microalga. It has been foundthat for certain microalga, a plant fatty acid synthesis gene product isfunctional in the absence of the corresponding plant acyl carrierprotein (ACP), even when the gene product is an enzyme, such as anacyl-ACP thioesterase, that requires binding of ACP to function. Thus,optionally, the microalgal cells can utilize such genes to make adesired oil without co-expression of the plant ACP gene.

III. Fatty Acid Auxotrophs/Reducing Fatty Acid Levels to GrowthInhibitory Conditions During an Oil Production Phase

In an embodiment, the cell isgenetically engineered so that all allelesof a lipid pathway gene are knocked out. Alternately, the amount oractivity of the gene products of the alleles is knocked down so as torequire supplementation with fatty acids. A first transformationconstruct can be generated bearing donor sequences homologous to one ormore of the alleles of the gene. This first transformation construct maybe introduced and selection methods followed to obtain an isolatedstrain characterized by one or more allelic disruptions. Alternatively,a first strain may be created that is engineered to express a selectablemarker from an insertion into a first allele, thereby inactivating thefirst allele. This strain may be used as the host for still furthergenetic engineering to knockout or knockdown the remaining allele(s) ofthe lipid pathway gene. Complementation of the endogenous gene can beachieved through engineered expression of an additional transformationconstruct bearing the endogenous gene whose activity was originallyablated, or through the expression of a suitable heterologous gene. Theexpression of the complementing gene can either be regulatedconstitutively or through regulatable control, thereby allowing fortuning of expression to the desired level so as to permit growth orcreate an auxotrophic condition at will. In an embodiment, a populationof the fatty acid auxotroph cells are used to screen or select forcomplementing genes; e.g., by transformation with particular genecandidates for exogenous fatty acid synthesis enzymes, or a nucleic acidlibrary believed to contain such candidates.

Knockout of all alleles of the desired gene and complementation of theknocked-out gene need not be carried out sequentially. The disruption ofan endogenous gene of interest and its complementation either byconstitutive or inducible expression of a suitable complementing genecan be carried out in several ways. In one method, this can be achievedby co-transformation of suitable constructs, one disrupting the gene ofinterest and the second providing complementation at a suitable,alternative locus. In another method, ablation of the target gene can beeffected through the direct replacement of the target gene by a suitablegene under control of an inducible promoter. In this way, expression ofthe targeted gene is now put under the control of a regulatablepromoter. An additional approach is to replace the endogenous regulatoryelements of a gene with an exogenous, inducible gene expression system.Under such a regime, the gene of interest can now be turned on or offdepending upon the particular needs. A still further method is to createa first strain to express an exogenous gene capable of complementing thegene of interest, then to knockout out or knockdown all alleles of thegene of interest in this first strain. The approach of multiple allelicknockdown or knockout and complementation with exogenous genes may beused to alter the fatty acid profile, regiospecific profile, sn-2profile, or the TAG profile of the engineered cell.

In a specific embodiment, the recombinant cell comprises nucleic acidsoperable to reduce the activity of an endogenous acyl-ACP thioesterase;for example a FatA or FatB acyl-ACP thioesterase having a preference forhydrolyzing fatty acyl-ACP chains of length C18 (e.g., stearate (C18:0)or oleate (C18:1), or C8:0-C16:0 fatty acids. The activity of anendogenous acyl-ACP thioesterase may be reduced by knockout or knockdownapproaches. Knockdown may be achieved through the use of one or more RNAhairpin constructs, by promoter hijacking (substitution of a loweractivity or inducible promoter for the native promoter of an endogenousgene), or by a gene knockout combined with introduction of a similar oridentical gene under the control of an inducible promoter. Example 34describes the engineering of a Prototheca strain in which two alleles ofthe endogenous fatty acyl-ACP thioesterase (FATA1) have been knockedout. The activity of the Prototheca moriformis FATA1 was complemented bythe expression of an exogenous thioesterase. Example 36 details the useof RNA hairpin constructs to reduce the expression of FATA1 inPrototheca.

Accordingly, oleaginous cells, including those of organisms with a typeII fatty acid biosynthetic pathway can have knockouts or knockdowns ofacyl-ACP-thioesterase encoding alleles to such a degree as to eliminateor severely limit viability of the cells in the absence of fatty acidsupplementation or genetic complementations. These strains can be usedto select for transformants expressing acyl-ACP-thioesterase transgenes.Alternately, or in addition, the strains can be used to completelytransplant exogenous acyl-ACP-thioesterases to give dramaticallydifferent fatty acid profiles of natural oils produced by such cells.For example, FATA expression can be completely or nearly completelyeliminated and replaced with FATB genes that produce mid-chain fattyacids. In specific embodiments, these transformants produce natural oilswith more than 50, 60, 70, 80, or 90% caprylic, capric, lauric,myristic, or palmitic acid, or total fatty acids of chain length lessthan 18 carbons. Such cells may require supplementation with longerchain fatty acids such as stearatic or oleic acid or switching ofenvironmental conditions between growth permissive and restrictivestates in the case of an inducible promoter regulating a FatA gene.

In an embodiment the oleaginous cells are cultured. The cells are fullyauxotrophic or partially auxotrophic (i.e., lethality or syntheticsickness) with respect to one or more types of fatty acid. The cells arecultured with supplementation of the fatty acid(s) so as to increase thecell number, then allowing the cells to accumulate oil (e.g. to at least40% by dry cell weight). Alternatively, the cells comprise a regulatablefatty acid synthesis gene that can be switched in activity based onenvironmental conditions and the environmental conditions during afirst, cell division, phase favor production of the fatty acid and theenvironmental conditions during a second, oil accumulation, phasedisfavor production of the fatty acid. In the case of an inducible gene,the regulation of the inducible gene can be mediated, withoutlimitation, via environmental pH (for example, by using the AMT3promoter described in the Examples).

As a result of applying either of these supplementation or regulationmethods, a cell oil may be obtained from the cell that has low amountsof one or more fatty acids essential for optimal cell propagation.Specific examples of oils that can be obtained include those low instearic, linoleic and/or linolenic acids.

These cells and methods are illustrated in connection with lowpolyunsaturated oils in the section immediately below and in Example 6(fatty acid desaturase auxotroph) in connection with oils low inpolyunsaturated fatty acids and in Example 34 (acyl-ACP thioesteraseauxotroph).

Likewise, fatty acid auxotrophs can be made in other fatty acidsynthesis genes including those encoding a SAD, FAD, KASIII, KASI,KASII, KCS, elongase, GPAT, LPAAT, DGAT or AGPAT or PAP. Theseauxotrophs can also be used to select for complement genes or toeliminate native expression of these genes in favor of desired exogenousgenes in order to alter the fatty acid profile, regiospecific profile,or TAG profile of natural oils produced by oleaginous cells.

Accordingly, in an embodiment of the invention, there is a method forproducing an oil/fat. The method comprises cultivating a recombinantoleaginous cell in a growth phase under a first set of conditions thatis permissive to cell division so as to increase the number of cells dueto the presence of a fatty acid, cultivating the cell in an oilproduction phase under a second set of conditions that is restrictive tocell division but permissive to production of an oil that is depleted inthe fatty acid, and extracting the oil from the cell, wherein the cellhas a mutation or exogenous nucleic acids operable to suppress theactivity of a fatty acid synthesis enzyme, the enzyme optionally being astearoyl-ACP desaturase, delta 12 fatty acid desaturase, or aketoacyl-ACP synthase. The oil produced by the cell can be depleted inthe fatty acid by at least than 50, 60, 70, 80, or 90%. The cell can becultivated heterotrophically. The cell can be a microalgal cell and mayproduce at least 40, 50, 60, 70, 80, or 90% oil by dry cell weight.

IV. Low Polyunsaturated Natural Oils

In an embodiment of the present invention, the natural oil produced bythe cell has very low levels of polyunsaturated fatty acids. As aresult, the natural oil can have improved stability, including oxidativestability. The natural oil can be a liquid or solid at room temperature,or a blend of liquid and solid oils, including the regiospecific orstererospecific oils, high stearate oils, or high mid-chain oilsdescribed infra. Oxidative stability can be measured by the Rancimatmethod using the AOCS Cd 12b-92 standard test at a defined temperature.For example, the OSI (oxidative stability index) test may be run attemperatures between 110° C. and 140° C. The oil is produced bycultivating cells (e.g., any of the plastidic microbial cells mentionedabove or elsewhere herein) that are genetically engineered to reduce theactivity of one or more fatty acid desaturase. For example, the cellsmay be genetically engineered to reduce the activity of one or morefatty acyl Δ12 desaturase(s) responsible for converting oleic acid(18:1) into linoleic acid (18:2) and/or one or more fatty acyl 415desaturase(s) responsible for converting linoleic acid (18:2) intolinolenic acid (18:3). Various methods may be used to inhibit thedesaturase including knockout or mutation of one or more alleles of thegene encoding the desaturase in the coding or regulatory regions,inhibition of RNA transcription, or translation of the enzyme, includingRNAi, siRNA, miRNA, dsRNA, antisense, and hairpin RNA techniques. Othertechniques known in the art can also be used including introducing anexogenous gene that produces an inhibitory protein or other substancethat is specific for the desaturase.

In a specific embodiment, fatty acid desaturase (e.g., Δ12 fatty aciddesaturase) activity in the cell is reduced to such a degree that thecell is unable to be cultivated or is difficult to cultivate (e.g., thecell division rate is decreased more than 10, 20, 30, 40, 50, 60, 70,80, 90, 95, 97 or 99%). Achieving such conditions may involve knockout,or effective suppression of the activity of multiple gene copies (e.g.2, 3, 4 or more) of the desaturase or their gene products. A specificembodiment includes the cultivation in cell culture of a full or partialfatty acid auxotroph with supplementation of the fatty acid or a mixtureof fatty acids so as to increase the cell number, then allowing thecells to accumulate oil (e.g. to at least 40% by cell weight).Alternatively, the cells comprise a regulatable fatty acid synthesisgene that can be switched in activity. For example, the regulation canbe based on environmental conditions and the environmental conditionsduring a first, cell division, phase favor production of the fatty acidand the environmental conditions during a second, oil accumulation,phase disfavor production of the oil. Examples of such cells aredescribed in Example 7.

In a specific embodiment, a cell is cultivated using a modulation oflinoleic acid levels within the cell. In particular, the natural oil isproduced by cultivating the cells under a first condition that ispermissive to an increase in cell number due to the presence of linoleicacid and then cultivating the cells under a second condition that ischaracterized by linoleic acid starvation and thus is inhibitory to celldivision, yet permissive of oil accumulation. For example, a seedculture of the cells may be produced in the presence of linoleic acidadded to the culture medium. For example, the addition of linoleic acidto 0.25 g/L in the seed culture of a Prototheca strain deficient inlinoleic acid production due to ablation of two alleles of a fatty acylΔ12 desaturase (i.e., a linoleic auxotroph) was sufficient to supportcell division to a level comparable to that of wild type cells.Optionally, the linoleic acid can then be consumed by the cells, orotherwise removed or diluted. The cells are then switched into an oilproducing phase (e.g., supplying sugar under nitrogen limitingconditions as described in WO2010/063032). Surprisingly, oil productionhas been found to occur even in the absence of linoleic acid, asdemonstrated in the obligate heterotroph oleaginous microalgaePrototheca but generally applicable to other oleaginous microalgae,microorganism, or even multicellular organisms (e.g., cultured plantcells). Under these conditions, the oil content of the cell can increaseto about 10, 20, 30, 40, 50, 60, 70, 80, 90%, or more by dry cellweight, while the oil produced can have polyunsaturated fatty acid(e.g.; linoleic+linolenic) profile with 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%,0.2%, 0.1%, 0.05% or less, as a percent of total triacylglycerol fattyacids in the oil. For example, the oil content of the cell can be 50% ormore by dry cell weight and the triglyceride of the oil produced lessthan 3% polyunsaturated fatty acids.

These oils can also be produced without the need (or reduced need) tosupplement the culture with linoleic acid by using cell machinery toproduce the linoleic acid, but predominantly or only during the celldivision phase. The linoleic-producing cell machinery may be regulatableso as to produce substantially less linoleic acid during the oilproducing phase. The regulation may be via modulation of transcriptionof the desaturase gene(s). For example, the majority, and preferablyall, of the fatty acid Δ12 desaturase activity can be placed under aregulatable promoter regulated to express the desaturase in the celldivision phase, but to be reduced or turned off during the oilaccumulation phase. The regulation can be linked to a cell culturecondition such as pH, and/or nitrogen level, as described in theexamples herein, or other environmental condition. In practice, thecondition may be manipulated by adding or removing a substance (e.g.,protons via addition of acid or base) or by allowing the cells toconsume a substance (e.g, nitrogen-supplying nutrients) to effect thedesired switch in regulation of the desaturase activity.

Other genetic or non-genetic methods for regulating the desaturaseactivity can also be used. For example, an inhibitor of the desaturasecan be added to the culture medium in a manner that is effective toinhibit polyunsaturated fatty acids from being produced during the oilproduction phase.

Using one or more of these desaturase regulation methods, it is possibleto obtain a natural oil that it is believed has been previouslyunobtainable, especially in large scale cultivation in a bioreactor(e.g., more than 1000 L). The oil can have polyunsaturated fatty acidlevels that are 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, 0.2%, or less, as apercent of total triacylglycerol fatty acids in the oil.

One consequence of having such low levels of polyunsaturates is thatoils are exceptionally stable to oxidation. Indeed, in some cases theoils may be more stable than any previously known natural cell oil. Inspecific embodiments, the oil is stable, without added antioxidants, at110° C. so that the inflection point in conductance is not yet reachedby 10 hours, 15 hours, 20 hours, 30 hours, 40, hours, 50 hours, 60hours, or 70 hours under conditions of the AOCS Cd 12b-92. Rancimattest, noting that for very stable oils, replenishment of water may berequired in such a test due to evaporation that occurs with such longtesting periods (see Example 5). For example the oil can have and OSIvalue of 40-50 hours or 41-46 hours at 110° C. without addedantioxidants. When antioxidants (suitable for foods or otherwise) areadded, the OSI value measured may be further increased. For example,with added tocopherol (100 ppm) and ascorbyl palmitate (500 ppm) or PANAand ascorbyl palmitate, such an oil can have an oxidative stabilityindex (OSI value) at 110° C. in excess 100 or 200 hours, as measured bythe Rancimat test. In another example, 1050 ppm of mixed tocopherols and500 pm of ascorbyl palmitate are added to an oil comprising less than 1%linoleic acid or less than 1% linoleic+linolenic acids; as a result, theoil is stable at 110° C. for 1, 2, 3, 4, 5, 6, 7, 8, or 9, 10, 11, 12,13, 14, 15, or 16, 20, 30, 40 or 50 days, 5 to 15 days, 6 to 14 days, 7to 13 days, 8 to 12 days, 9 to 11 days, about 10 days, or about 20 days.In a further embodiment, the OSI value of the natural oil without addedantioxidants at 120° C. is greater than 15 hours or 20 hours or is inthe range of 10-15, 15-20, 20-25, or 25-50 hours.

In an example, using these methods, the oil content of a microalgal cellis between 40 and about 85% by dry cell weight and the polyunsaturatedfatty acids in the fatty acid profile of the oil is between 0.001% and3% in the fatty acid profile of the oil and optionally yields a naturaloil having an OSI induction time of at least 20 hours at 110° C. withoutthe addition of antioxidants. In yet another example, there is a naturaloil produced by RBD treatment of a natural oil from an oleaginous cell,the oil comprises between 0.001% and 2% polyunsaturated fatty acids andhas an OSI induction time exceeding 30 hours at 110 C without theaddition of antioxidants. In yet another example, there is a natural oilproduced by RBD treatment of a natural oil from an oleaginous cell, theoil comprises between 0.001% and 1% polyunsaturated fatty acids and hasan OSI induction time exceeding 30 hours at 110 C without the additionof antioxidants.

In another specific embodiment there is an oil with reducedpolyunsaturate levels produced by the above-described methods. The oilis combined with antioxidants such as PANA and ascorbyl palmitate. Forexample, it was found that when such an oil was combined with 0.5% PANAand 500 ppm of ascorbyl palmitate the oil had an OSI value of about 5days at 130° C. or 21 days at 110° C. These remarkable results suggestthat not only is the oil exceptionally stable, but these twoantioxidants are exceptionally potent stabilizers of triglyceride oilsand the combination of these antioxidants may have general applicabilityincluding in producing stable biodegradable lubricants (e.g., jet enginelubricants). In specific embodiments, the genetic manipulation of fattyacyl Δ12 desaturase results in a 2 to 30, or 5 to 25, or 10 to 20 foldincrease in OSI (e.g., at 110° C.) relative to a strain without themanipulation. The oil can be produced by suppressing desaturase activityin a cell, including as described above.

Antioxidants suitable for use with the oils of the present invention mayinclude alpha, delta, and gamma tocopherol (vitamin E), tocotrienol,ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid,β-carotene, lycopene, lutein, retinol (vitamin A), ubiquinol (coenzymeQ), melatonin, resveratrol, flavonoids, rosemary extract, propyl gallate(PG), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA),and butylated hydroxytoluene (BHT),N,N′-di-2-butyl-1,4-phenylenediamine,2,6-di-tert-butyl-4-methylphenol,2,4-dimethyl-6-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol,2,4-dimethyl-6-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol,2,6-di-tert-butylphenol, and phenyl-alpha-naphthylamine (PANA).

In addition to the desaturase modifications, in a related embodimentother genetic modifications may be made to further tailor the propertiesof the oil, as described throughout, including introduction orsubstitution of acyl-ACP thioesterases having altered chain lengthspecificity and/or overexpression of an endogenous or exogenous geneencoding a KAS, SAD, LPAAT, or DGAT gene. For example, a strain thatproduces elevated oleic levels may also produce low levels ofpolyunsaturates. Such genetic modifications can include increasing theactivity of stearoyl-ACP desaturase (SAD) by introducing an exogenousSAD gene, increasing elongase activity by introducing an exogenous KASIIgene, and/or knocking down or knocking out a FATA gene.

In a specific embodiment, a high oleic natural oil with lowpolyunsaturates may be produced. For example, the oil may have a fattyacid profile with greater than 60, 70, 80, 90, or 95% oleic acid andless than 5, 4, 3, 2, or 1% polyunsaturates. In related embodiments, anatural oil is produced by a cell having recombinant nucleic acidsoperable to decrease fatty acid Δ12 desaturase activity and optionallyfatty acid 415 desaturase so as to produce an oil having less than orequal to 3% polyunsaturated fatty acids with greater than 60% oleicacid, less than 2% polyunsaturated fatty acids and greater than 70%oleic acid, less than 1% polyunsaturated fatty acids and greater than80% oleic acid, or less than 0.5% polyunsaturated fatty acids andgreater than 90% oleic acid. It has been found that one way to increaseoleic acid is to use recombinant nucleic acids operable to decreaseexpression of an acyl-ACP thioesterase and optionally overexpress a KASII gene; such a cell can produce an oil with greater than or equal to75% oleic acid. Thus, the oil produced can have a fatty acid profilewith at least 75% oleic and at most 3%, 2%, 1, or 0.5% linoleic acid. Ina related example, the oil has between 80 to 95% oleic acid and about0.001 to 2% linoleic acid, 0.01 to 2% linoleic acid, or 0.1 to 2%linoleic acid. Such oils will have a low freezing point, with excellentstability and are useful in foods, for frying, fuels, or in chemicalapplications. Further, these oils may exhibit a reduced propensity tochange color over time. In an illustrative chemical application, thehigh oleic oil is used to produce a chemical. The oleic acid doublebonds of the oleic acid groups of the triglycerides in the oil can beepoxidized or hydroxylated to make a polyol. The epoxidized orhydroxylated oil can be used in a variety of applications. One suchapplication is the production of polyurethane (including polyurethanefoam) via condensation of the hydroxylated triglyceride with anisocyante, as has been practiced with hydroxylated soybean oil or castoroil. See, e.g. US2005/0239915, US2009/0176904, US2005/0176839,US2009/0270520, and U.S. Pat. No. 4,264,743 and Zlatanic, et al,Biomacromolecules 2002, 3, 1048-1056 (2002) for examples ofhydroxylation and polyurethane condensation chemistries. Suitablehydroxyl forming reactions include epoxidation of one or more doublebonds of a fatty acid followed by acid catalyzed epoxide ring openingwith water (to form a diol), alcohol (to form a hydroxyl ether), or anacid (to form a hydroxyl ester). There are multiple advantages of usingthe high-oleic/low polyunsaturated oil in producing a bio-basedpolyurethane: (1) the shelf-life, color or odor, of polyurethane foamsmay be improved; (2) the reproducibility of the product may be improveddue to lack of unwanted side reactions resulting from polyunsaturates;(3) a greater degree of hydroxylation reaction may occur due to lack ofpolyunsaturates and the structural characteristics of the polyurethaneproduct can be improved accordingly.

The low polyunsaturated or high oleic/low polyunsaturated oils describedhere may be advantageously used in chemical applications where yellowingis undesirable. For example, yellowing can be undesirable in paints orcoatings made from the triglycerides fatty acids derived from thetriglycerides. Yellowing may be caused by reactions involvingpolyunsaturated fatty acids and tocotrienols and/or tocopherols. Thus,producing the high stability oil in an oleaginous microbe with lowlevels of tocotrienols can be advantageous in elevating high colorstability a chemical composition made using the oil. In contrast tocommonly used plant oils, through appropriate choice of oleaginousmicrobe, the natural oils of these embodiments can have tocopherols andtocotrienols levels of 1 g/L or less. In a specific embodiment, anatural oil has a fatty acid profile with less than 2% withpolyunsaturated fatty acids and less than 1 g/L for tocopherols,tocotrienols or the sum of tocopherols and tocotrienols. In anotherspecific embodiment, the natural oil has a fatty acid profile with lessthan 1% with polyunsaturated fatty acids and less than 0.5 g/L fortocopherols, tocotrienols or the sum of tocopherols and tocotrienols

Any of the high-stability (low-polyunsaturate) natural oils orderivatives thereof can be used to formulate foods, drugs, vitamins,nutraceuticals, personal care or other products, and are especiallyuseful for oxidatively sensitive products. For example, thehigh-stability natural oil (e.g., less than or equal to 3%, 2% or 1%polyunsaturates) can be used to formulate a sunscreen (e.g. acomposition having one or more of avobenzone, homosalate, octisalate,octocrylene or oxybenzone) or retinoid face cream with an increasedshelf life due to the absence of free-radical reactions associated withpolyunsaturated fatty acids. For example, the shelf-life can beincreased in terms of color, odor, organoleptic properties or % activecompound remaining after accelerated degradation for 4 weeks at 54° C.The high stability oil can also be used as a lubricant with excellenthigh-temperature stability. In addition to stability, the oils can bebiodegradable, which is a rare combination of properties.

In another related embodiment, the fatty acid profile of a natural oilis elevated in C8 to C16 fatty acids through additional geneticmodification, e.g. through overexpression of a short-chain to mid chainpreferring acyl-ACP thioesterase or other modifications described here.A low polyunsaturated oil in accordance with these embodiments can beused for various industrial, food, or consumer products, including thoserequiring improved oxidative stability. In food applications, the oilsmay be used for frying with extended life at high temperature, orextended shelf life.

Where the oil is used for frying, the high stability of the oil mayallow for frying without the addition of antioxidant and/or defoamers(e.g. silicone). As a result of omitting defoamers, fried foods mayabsorb less oil. Where used in fuel applications, either as atriglyceride or processed into biodiesel or renewable diesel (see, e.g.,WO2008/151149 WO2010/063032, and WO2011/150410), the high stability canpromote storage for long periods, or allow use at elevated temperatures.For example, the fuel made from the high stability oil can be stored foruse in a backup generator for more than a year or more than 5 years. Thefrying oil can have a smoke point of greater than 200° C., and freefatty acids of less than 0.1%.

The low polyunsaturated oils may be blended with food oils, includingstructuring fats such as those that form beta or beta prime crystals,including those produced as described below. These oils can also beblended with liquid oils. If mixed with an oil having linoleic acid,such as corn oil, the linoleic acid level of the blend may approximatethat of high oleic plant oils such as high oleic sunflower oils (e.g.,about 80% oleic and 8% linoleic).

Blends of the low polyunsaturated natural oil can be interesterifiedwith other oils. For example, the oil can be chemically or enzymaticallyinteresterified. In a specific embodiment, a low polyunsaturated oilaccording to an embodiment of the invention has at least 10% oleic acidin its fatty acid profile and less than 5% polyunsaturates and isenzymatically interesterified with a high saturate oil (e.g.hydrogenated soybean oil or other oil with high stearate levels) usingan enzyme that is specific for sn-1 and sn-2 triacylglycerol positions.The result is an oil that includes a stearate-oleate-stearate (SOS).Methods for interesterification are known in the art; see for example,“Enzymes in Lipid Modification,” Uwe T. Bornschuer, ed., Wiley_VCH,2000, ISBN 3-527-30176-3.

V. Cells with Exogenous Acyltransferases

In various embodiments of the present invention, one or more genesencoding an acyltransferase (an enzyme responsible for the condensationof a fatty acid with glycerol or a glycerol derivative to form anacylglyceride) can be introduced into an oleaginous cell (e.g., aplastidic microalgal cell) so as to alter the fatty acid composition ofa natural oil produced by the cell. The genes may encode one or more ofa glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acidacyltransferase (LPAAT), also known as 1-acylglycerol-3-phosphateacyltransferase (AGPAT), phosphatidic acid phosphatase (PAP), ordiacylglycerol acyltransferase (DGAT) that transfers an acyl group tothe sn-3 position of DAG, thereby producing a TAG.

Recombinant nucleic acids may be integrated into a plasmid or chromosomeof the cell. Alternately, the gene encodes an enzyme of a lipid pathwaythat generates TAG precursor molecules through fattyacyl-CoA-independent routes separate from that above. Acyl-ACPs may besubstrates for plastidial GPAT and LPAAT enzymes and/or mitochondrialGPAT and LPAAT enzymes. Among further enzymes capable of incorporatingacyl groups (e.g., from membrane phospholipids) to produce TAGs isphospholipid diacylglycerol acyltransferase (PDAT). Still furtheracyltransferases, including lysophosphosphatidylcholine acyltransferase(LPCAT), lysophosphosphatidylserine acyltransferase (LPSAT),lysophosphosphatidylethanolamine acyltransferase (LPEAT), andlysophosphosphatidylinositol acyltransferase (LPIAT), are involved inphospholipid synthesis and remodeling that may impact triglyceridecomposition.

The exogenous gene can encode an acyltransferase enzyme havingpreferential specificity for transferring an acyl substrate comprising aspecific number of carbon atoms and/or a specific degree of saturationis introduced into a oleaginous cell so as to produce an oil enriched ina given regiospecific triglyceride. For example, the coconut (Cocosnucifera) lysophosphatidic acid acyltransferase has been demonstrated toprefer C12:0-CoA substrates over other acyl-CoA substrates (Knutzon etal., Plant Physiology, Vol. 120, 1999, pp 739-746), whereas the1-acyl-sn-3-glycerol-3-phosphate acyltransferase of maturing safflowerseeds shows preference for linoleoyl-CoA and oleyl-CoA substrates overother acyl-CoA substrates, including stearoyl-CoA (Ichihara et al.,European Journal of Biochemistry, Vol. 167, 1989, pp 339-347).Furthermore, acyltransferase proteins may demonstrate preferentialspecificity for one or more short-chain, medium-chain, or long-chainacyl-CoA or acyl-ACP substrates, but the preference may only beencountered where a particular, e.g. medium-chain, acyl group is presentin the sn-1 or sn-3 position of the lysophosphatidic acid donorsubstrate. As a result of the exogenous gene, a TAG oil can be producedby the cell in which a particular fatty acid is found at the sn-2position in greater than 20, 30, 40, 50, 60, 70, 90, or 90% of the TAGmolecules.

In some embodiments of the invention, the cell makes an oil rich insaturated-unsaturated-saturated (sat-unsat-s at) TAGs. Sat-unsat-s atTAGS include 1,3-dihexadecanoyl-2-(9Z-octadecenoyl)-glycerol (referredto as 1-palmitoyl-2-oleyl-glycero-3-palmitoyl),1,3-dioctadecanoyl-2-(9Z-octadecenoyl)-glycerol (referred to as1-stearoyl-2-oleyl-glycero-3-stearoyl), and1-hexadecanoyl-2-(9Z-octadecenoyl)-3-octadecanoy-glycerol (referred toas 1-palmitoyl-2-oleyl-glycero-3-stearoyl). These molecules are morecommonly referred to as POP, SOS, and POS, respectively, where ‘P’represents palmitic acid, ‘S’ represents stearic acid, and ‘O’represents oleic acid. Further examples ofsaturated-unsaturated-saturated TAGs include MOM, LOL, MOL, COC and COL,where ‘M’ represents myristic acid, 1′ represents lauric acid, and ‘C’represents capric acid (C8:0). Trisaturates, triglycerides with threesaturated fatty acyl groups, are commonly sought for use in foodapplications for their greater rate of crystallization than other typesof triglycerides. Examples of trisaturates include PPM, PPP, LLL, SSS,CCC, PPS, PPL, PPM, LLP, and LLS. In addition, the regiospecificdistribution of fatty acids in a TAG is an important determinant of themetabolic fate of dietary fat during digestion and absorption.

According to certain embodiments of the present invention, oleaginouscells are transformed with recombinant nucleic acids so as to producenatural oils that comprise an elevated amount of a specifiedregiospecific triglyceride, for example 1-acyl-2-oleyl-glycero-3-acyl,or 1-acyl-2-lauric-glycero-3-acyl where oleic or lauric acidrespectively is at the sn-2 position, as a result of introducedrecombinant nucleic acids. Alternately, caprylic, capric, myristic, orpalmitic acid may be at the sn-2 position. The amount of the specifiedregiospecific triglyceride present in the natural oil may be increasedby greater than 5%, greater than 10%, greater than 15%, greater than20%, greater than 25%, greater than 30%, greater than 35%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90%, greater than 100-500%, or greater than 500% thanin the natural oil produced by the microorganism without the recombinantnucleic acids. As a result, the sn-2 profile of the cell triglyceridemay have greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90% of theparticular fatty acid.

The identity of the acyl chains located at the distinct stereospecificor regiospecific positions in a glycerolipid can be evaluated throughone or more analytical methods known in the art (see Luddy et al., J.Am. Oil Chem. Soc., 41, 693-696 (1964), Brockerhoff, J. Lipid Res., 6,10-15 (1965), Angers and Aryl, J. Am. Oil Chem. Soc., Vol. 76:4, (1999),Buchgraber et al., Eur. J. Lipid Sci. Technol., 106, 621-648 (2004)), orin accordance with Examples 1, 2, and 8 given below.

The positional distribution of fatty acids in a triglyceride moleculecan be influenced by the substrate specificity of acyltransferases andby the concentration and type of available acyl moieties. Nonlimitingexamples of enzymes suitable for altering the regiospecificity of atriglyceride produced in a recombinant microorganism are listed inTables 1-4. One of skill in the art may identify additional suitableproteins.

TABLE 1 Glycerol-3-phosphate acyltransferases and GenBank accessionnumbers. glycerol-3-phosphate acyltransferase Arabidopsis BAA00575thaliana glycerol-3-phosphate acyltransferase Chlamydomonas EDP02129reinhardtii glycerol-3-phosphate acyltransferase Chlamydomonas Q886Q7reinhardtii acyl-(acyl-carrier-protein): Cucurbita moschata BAB39688glycerol-3-phosphate acyltransferase glycerol-3-phosphateacyltransferase Elaeis guineensis AAF64066 glycerol-3-phosphateacyltransferase Garcina ABS86942 mangostana glycerol-3-phosphateacyltransferase Gossypium hirsutum ADK23938 glycerol-3-phosphateacyltransferase Jatropha curcas ADV77219 plastid glycerol-3-phosphateJatropha curcas ACR61638 acyltransferase plastidial glycerol-phosphateRicinus communis EEF43526 acyltransferase glycerol-3-phosphateacyltransferase Vica faba AAD05164 glycerol-3-phosphate acyltransferaseZea mays ACG45812

Lysophosphatidic acid acyltransferases suitable for use with themicrobes and methods of the invention include, without limitation, thoselisted in Table 2.

TABLE 2 Lysophosphatidic acid acyltransferases and GenBank accessionnumbers. 1-acyl-sn-glycerol-3-phosphate Arabidopsis thaliana AEE85783acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica juncea ABQ42862acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica juncea ABM92334acyltransferase 1-acyl-sn-glycerol-3-phosphate Brassica napus CAB09138acyltransferase lysophosphatidic acid Chlamydomonas EDP02300acyltransferase reinhardtii lysophosphatidic acid Limnanthes albaAAC49185 acyltransferase 1-acyl-sn-glycerol-3-phosphate Limnanthesdouglasii CAA88620 acyltransferase (putative)acyl-CoA:sn-1-acylglycerol-3- Limnanthes douglasii ABD62751 phosphateacyltransferase 1-acylglycerol-3-phosphate Limnanthes douglasii CAA58239O-acyltransferase 1-acyl-sn-glycerol-3-phosphate Ricinus communisEEF39377 acyltransferase

Diacylglycerol acyltransferases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 3.

TABLE 3 Diacylglycerol acyltransferases and GenBank accession numbers.diacylglycerol acyltransferase Arabidopsis CAB45373 thalianadiacylglycerol acyltransferase Brassica juncea AAY40784 putativediacylglycerol Elaeis guineensis AEQ94187 acyltransferase putativediacylglycerol Elaeis guineensis AEQ94186 acyltransferase acylCoA:diacylglycerol Glycine max AAT73629 acyltransferase diacylglycerolacyltransferase Helianthus annus ABX61081 acyl-CoA:diacylglycerol Oleaeuropaea AAS01606 acyltransferase 1 diacylglycerol acyltransferaseRicinus communis AAR11479

Phospholipid diacylglycerol acyltransferases suitable for use with themicrobes and methods of the invention include, without limitation, thoselisted in Table 4.

TABLE 4 Phospholipid diacylglycerol acyltransferases and GenBankaccession numbers. phospholipid:diacylglycerol Arabidopsis AED91921acyltransferase thaliana putativephospholipid:diacylglycerol ElaeisAEQ94116 acyltransferase guineensis phospholipid:diacylglycerol Glycinemax XP_003541296 acyltransferase 1-like phospholipid:diacylglycerolJatropha AEZ56255 acyltransferase curcas phospholipid:diacylglycerolRicinus ADK92410 acyltransferase communis phospholipid:diacylglycerolRicinus AEW99982 acyltransferase communis

In embodiment of the invention, known or novel LPAAT genes aretransformed into the oleaginous cells so as to alter the fatty acidprofile of triglycerides produced by those cells, most notably byaltering the sn-2 profile of the triglycerides. For example, by virtueof expressing an exogenous active LPAAT in an oleaginous cell, thepercent of unsaturated fatty acid at the sn-2 position is increased by10, 20, 30, 40, 50, 60, 70, 80, 90% or more. For example, a cell mayproduce triglycerides with 30% unsaturates (which may be primarily 18:1and 18:2 and 18:3 fatty acids) at the sn-2 position. In this example,introduction of the LPAAT activity increases the unsaturates at the sn-2position by 20% so that 36% of the triglycerides comprise unsaturates atthe sn-2 position. Alternately, an exogenous LPAAT can be used toincrease mid-chain fatty acids including saturated mid-chains such asC8:0, C10:0, C12:0, C14:0 or C16:0 moieties at the sn-2 position. As aresult, mid-chain levels in the overall fatty acid profile may beincreased. Examples 43 and 44 describe altering the sn-2 and fatty acidprofiles in an oleaginous microbe. As can be seen from those examples,the choice of LPAAT gene is important in that different LPAATs can causea shift in the sn-2 and fatty acid profiles toward differentchain-lengths. For example, the LPAAT of Example 43 increases C10-C14fatty acids and the LPAAT of Example 44 causes an increase in C16 andC18 fatty acids. As in these examples, introduction of an exogenousLPAAT can be combined with introduction of exogenous acy-ACPthioesterase. Combining a mid-chain preferring LPAAT and a mid-chainpreferring FatB was found to give an additive effect; the fatty acidprofile was shifted more toward the mid-chain fatty acids more when bothan exogenous LPAAT and FatB gene was present than when only an exogenousFatB gene was present.

Specific embodiments of the invention are a nucleic acid construct, acell comprising the nucleic acid construct, a method of cultivating thecell to produce a triglyceride, and the triglyceride oil produced wherethe nucleic acid construct has a promoter operably linked to a novelLPAAT coding sequence. The coding sequence can have an initiation codonupstream and a termination codon downstream followed by a 3 UTRsequence. In a particular, specific embodiment, the LPAAT gene has acoding sequence have at least 80, 85, 90 or 95% sequence identity to thefollowing coding sequence obtained from Cuphea PSR23:

Atcgagcaggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgaccgcctgtccgcccagggccgccccgtgctgacgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccacgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgacgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcactccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcacctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccactaccgcctgc tggacgagacttc

VI. Cells with Exogenous Elongases or Elongase Complex Enzymes

In various embodiments of the present invention, one or more genesencoding elongases or components of the fatty acyl-CoA elongationcomplex can be introduced into an oleaginous cell (e.g., a plastidicmicroalgal cell) so as to alter the fatty acid composition of the cellor of a natural oil produced by the cell. The genes may encode abeta-ketoacyl-CoA synthase (also referred to as 3-ketoacyl synthase orKCS), a ketoacyl-CoA reductase, a hydroxyacyl-CoA dehydratase, enoyl-CoAreductase, or elongase. Recombinant nucleic acids may be integrated intoa plasmid or chromosome of the cell. In a specific embodiment, the cellis of Chlorophyta, including heterotrophic cells such as those of thegenus Prototheca.

Beta-Ketoacyl-CoA synthase and elongase enzymes suitable for use withthe microbes and methods of the invention include, without limitation,those listed in Table 5.

TABLE 5 Beta-Ketoacyl-CoA synthases and elongases listed with GenBankaccession numbers. Trypanosoma brucei elongase 3 (GenBank Accession No.AAX70673), Marchanita polymorpha (GenBank Accession No. AAP74370),Trypanosoma cruzi fatty acid elongase, putative (GenBank Accession No.EFZ33366), Nannochloropsis oculata fatty acid elongase (GenBankAccession No. ACV21066.1), Leishmania donovani fatty acid elongase,putative (GenBank Accession No. CBZ32733.1), Glycine max 3-ketoacyl- CoAsynthase 11-like (GenBank Accession No. XP_003524525.1), Medicagotruncatula beta-ketoacyl-CoA synthase (GenBank Accession No.XP_003609222), Zea mays fatty acid elongase (GenBank Accession No.ACG36525), Gossypium hirsutum beta-ketoacyl-CoA synthase (GenBankAccession No. ABV60087), Helianthus annuus beta-ketoacyl-CoA synthase(GenBank Accession No. ACC60973.1), Saccharomyces cerevisiae ELO1(GenBank Accession No. P39540), Simmondsia chinensis beta-ketoacyl-CoAsynthase (GenBank Accession No. AAC49186), Tropaeolum majus putativefatty acid elongase (GenBank Accession No. AAL99199, Brassica napusfatty acid elongase (GenBank Accession No. AAA96054)

In an embodiment of the invention, an exogenous gene encoding abeta-ketoacyl-CoA synthase or elongase enzyme having preferentialspecificity for elongating an acyl substrate comprising a specificnumber of carbon atoms and/or a specific degree of acyl chain saturationis introduced into a oleaginous cell so as to produce a cell or an oilenriched in fatty acids of specified chain length and/or saturation.Example 40 describes engineering of Prototheca strains in whichexogenous elongases with preferences for extending midchain fattyacyl-CoAs have been overexpressed to increase the concentration ofstearate. Example 42 describes engineering of Prototheca in whichexogenous elongases or beta-ketoacyl-CoA synthases with preferences forextending monounsaturated and saturated C18- and C20-CoA substrates areoverexpressed to increase the concentration of erucic acid.

VII. Regiospecific and Stereospecific Oils/Fats

In an embodiment, a recombinant cell produces a natural fat or oilhaving a given regiospecific makeup. As a result, the cell can producetriglyceride fats having a tendency to form crystals of a givenpolymorphic form; e.g., when heated to above melting temperature andthen cooled to below melting temperature of the fat For example, the fatmay tend to form crystal polymorphs of the β or β′ form (e.g., asdetermined by X-ray diffraction analysis), either with or withouttempering. The fats may be ordered fats. In specific embodiments, thefat may directly form either β or β′ crystals upon cooling;alternatively, the fat can proceed through a β form to a β′ form. Suchfats can be used as structuring laminating or coating fats for foodapplications. The natural fats can be incorporated into candy, dark orwhite chocolate, chocolate flavored confections, ice cream, margarinesor other spreads, cream fillings, pastries, or other food products.Optionally, the fats can be semisolid yet free of artificially producedtrans-fatty acids. Such fats can also be useful in skin care and otherconsumer or industrial products.

As in the other embodiments, the fat can be produced by geneticengineering of a plastidic cell, including heterotrophic microalgae ofthe phylum Chlorpophya, the class Trebouxiophytae, the orderChlorellales, or the family Chlorellacae. Preferably, the cell isoleaginous and capable of accumulating at least 40% oil by dry cellweight. The cell can be an obligate heterotroph, such as a species ofPrototheca, including Prototheca moriformis or Prototheca zopfii. Thefats can also be produced in autotrophic algae or plants. Optionally,the cell is capable of using sucrose to produce oil and a recombinantinvertase gene may be introduced to allow metabolism of sucrose, asdescribed in PCT Publications WO2008/151149, WO2010/06032,WO2011/150410, WO2011/150411, and international patent applicationPCT/US12/23696. The invertase may be codon optimized and integrated intoa chromosome of the cell, as may all of the genes mentioned here.

In an embodiment, the natural fat has at least 30, 40, 50, 60, 70, 80,or 90% fat of the general structure [saturated fatty acid(sn-1)-unsaturated fatty acid(sn-2)-saturated fatty acid(sn-3)]. This isdenoted below as Sat-Unsat-Sat fat. In a specific embodiment, thesaturated fatty acid in this structure is preferably stearate orpalmitate and the unsaturated fatty acid is preferably oleate. As aresult, the fat can form primarily β or β′ polymorphic crystals, or amixture of these, and have corresponding physical properties, includingthose desirable for use in foods or personal care products. For example,the fat can melt at mouth temperature for a food product or skintemperature for a cream, lotion or other personal care product (e.g., amelting temperature of 30 to 40, or 32 to 35° C.). Optionally, the fatscan have a 2L or 3L lamellar structure (e.g., as determined by X-raydiffraction analysis). Optionally, the fat can form this polymorphicform without tempering.

In a specific related embodiment, a natural fat triglyceride has a highconcentration of SOS (i.e. triglyceride with stearate at the terminalsn-1 and sn-3 positions, with oleate at the sn-2 position of theglycerol backbone). For example, the fat can have at least 50, 60, 70,80 or 90% SOS. In an embodiment, the fat has triglyceride of at least80% SOS. Optionally, at least 50, 60, 70, 80 or 90% of the sn-2 linkedfatty acids are unsaturated fatty acids. In a specific embodiment, atleast 95% of the sn-2 linked fatty acids are unsaturated fatty acids. Inaddition, the SSS (tri-stearate) level can be less than 20, 10 or 5%and/or the C20:0 fatty acid (arachidic acid) level may be less than 6%,and optionally greater than 1% (e.g., from 1 to 5%). For example, in aspecific embodiment, a natural fat produced by a recombinant cell has atleast 70% SOS triglyceride with at least 80% sn-2 unsaturated fatty acylmoieties. In another specific embodiment, a natural fat produced by arecombinant cell has TAGs with at least 80% SOS triglyceride and with atleast 95% sn-2 unsaturated fatty acyl moieties. In yet another specificembodiment, a natural fat produced by a recombinant cell has TAGs withat least 80% SOS, with at least 95% sn-2 unsaturated fatty acylmoieties, and between 1 to 6% C20 fatty acids.

In yet another specific embodiment, the sum of the percent stearate andpalmitate in the fatty acid profile of the natural fat is twice thepercentage of oleate, ±10, 20, 30 or 40% [e.g., (% P+% S)/% O=2.0±20%].Optionally, the sn-2 profile of this fat is at least 40%, and preferablyat least 50, 60, 70, or 80% oleate. Also optionally, this fat may be atleast 40, 50, 60, 70, 80, or 90% SOS. Optionally, the fat comprisesbetween 1 to 6% C20 fatty acids.

In any of these embodiments, the high SatUnsatSat fat may tend to formβ′ polymorphic crystals. Unlike previously available plant fats likecocoa butter, the SatUnsatSat fat produced by the cell may form β′polymorphic crystals without tempering. In an embodiment, the polymorphforms upon heating to above melting temperature and cooling to less thatthe melting temperature for 3, 2, 1, or 0.5 hours. In a relatedembodiment, the polymorph forms upon heating to above 60° C. and coolingto 10° C. for 3, 2, 1, or 0.5 hours.

In various embodiments the fat forms polymorphs of the β form, β′ form,or both, when heated above melting temperature and the cooled to belowmelting temperature, and optionally proceeding to at least 50% ofpolymorphic equilibrium within 5, 4, 3, 2, 1, 0.5 hours or less whenheated to above melting temperature and then cooled at 10° C. The fatmay form β crystals at a rate faster than that of cocoa butter.

Optionally, any of these fats can have less than 2 mole %diacylglycerol, or less than 2 mole % mono and diacylglycerols, in sum.

In an embodiment, the fat may have a melting temperature of between30-40° C., 32 to 37° C., 40 to 60° C. or 45 to 55° C. In anotherembodiment, the fat can have a solid fat content (SFC) of 40 to 50%, 15to 25%, or less than 15% at 20° C. and/or have an SFC of less than 15%at 35° C.

The cell used to make the fat may include recombinant nucleic acidsoperable to modify the saturate to unsaturate ratio of the fatty acidsin the cell triglyceride in order to favor the formation of SatUnsatSatfat. For example, a knock-out or knock-down of stearoyl-ACP desaturase(SAD) can be used to favor the formation of stearate over oleate orexpression of an exogenous mid-chain-preferring acyl-ACP thioesterasecan increase the levels mid-chain saturates. Alternately a gene encodinga SAD enzyme can be overexpressed to increase unsaturates.

In a specific embodiment, the cell has recombinant nucleic acidsoperable to elevate the level of stearate in the cell. As a result, theconcentration of SOS maybe increased. Example 9 demonstrates that theregiospecific profile of the recombinant microbe is enriched for POP,POS, and SOS TAGs as a result of overexpressing a Brassica napusC18:0-preferring thioesterase. An additional way to increase thestearate of a cell is to decrease oleate levels. For cells having higholeate levels (e.g., in excess of one half the stearate levels) one canalso employ recombinant nucleic acids or classical genetic mutationsoperable to decrease oleate levels. For example, the cell can have aknockout, knockdown, or mutation in one or more FATA alleles, whichencode an oleate liberating acyl-ACP thioesterase, and/or one or morealleles encoding a stearoyl ACP desaturase (SAD). Example 35 describesthe inhibition of SAD2 gene product expression using hairpin RNA toproduce a fatty acid profile of 37% stearate in Prototheca moriformis(UTEX 1435), whereas the wildtype strain produced less than 4% stearate,a more than 9-fold improvement. Moreover, while the strains of Example35 are engineered to reduce SAD activity, sufficient SAD activityremains to produce enough oleate to make SOS, POP, and POS. See the TAGprofiles of Example 47. In specific examples, one of multiple SADencoding alleles may be knocked out and/or one or more alleles aredownregulated using inhibition techniques such as antisense, RNAi, orsiRNA, hairpin RNA or a combination thereof. In various embodiments, thecell can produce TAGs that have 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, or 90 to about 100% stearate. In other embodiments, thecells can produce TAGs that are 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90, or 90 to about 100% SOS. Optionally, or in addition togenetic modification, stearoyl ACP desaturase can be inhibitedchemically; e.g., by addition of sterculic acid to the cell cultureduring oil production.

Surprisingly, knockout of a single FATA allele has been found toincrease the presence of C18 fatty acids produced in microalgae. Byknocking out one allele, or otherwise suppressing the activity of theFATA gene produce (e.g., using hairpin) RNA, while also suppressing theactivity of stearoyl-ACP desaturase (using techniques disclosed herein),stearate levels in the cell can be increased.

Another genetic modification to increase stearate levels includesincreasing a ketoacyl ACP synthase (KAS) activity in the cell so as toincrease the rate of stearate production. It has been found that inmicroalgae, increasing KASII activity is effective in increasing C18synthesis and particularly effective in elevating stearate levels incell triglyceride in combination with recombinant DNA effective indecreasing SAD activity.

Optionally, the cell can include an exogenous stearate liberatingacyl-ACP thioesterase, either as a sole modification or in combinationwith one or more other stearate-increasing genetic modifications. Forexample the cell be may engineered to overexpress an acyl-ACPthioesterase with preference for cleaving C18:0-ACPs. Example 9describes the expression of exogenous C18:0-preferring acyl-ACPthioesterases to increase stearate in the fatty acid profile ofPrototheca moriformis (UTEX 1435) from about 3.7% to about 30.4%.Example 41 provides additional examples of C18:0-preferring acyl-ACPthioesterases function to elevate C18:0 levels in Prototheca.Introduction of the thioesterase can be combined with a knockout orknockdown of one or more endogenous acyl-ACP thioesterase alleles.Introduction of the thioesterase can also be combined withoverexpression of an elonagase or beta-ketoacyl-CoA synthase. Inaddition, one or more exogenous genes (e.g., encoding SAD or KASII) canbe regulated via an environmental condition (e.g., by placement inoperable linkage with a regulatable promoter). In a specific example, pHand/or nitrogen level is used to regulate an amt03 promoter. Theenvironmental condition may then be modulated to tune the cell toproduce the desired amount of stearate appearing in cell triglycerides(e.g., to twice the oleate concentration). As a result of thesemanipulations, the cell may exhibit an increase in stearate of at least5, 10, 15, or 20 fold.

As a further modification alone or in combination with the otherstearate increasing modifications, the cell can comprise recombinantnucleic acids operable to express an elongase or a beta-ketoacyl-CoAsynthase. For example, overexpression of a C18:0-preferring acyl-ACPthioesterases may be combined with overexpression of amidchain-extending elongase or KCS to increase the production ofstearate in the recombinant cell. One or more of the exogenous genes(e.g., ending a thioesterase, elongase, or KCS) can be regulated via anenvironmental condition (e.g., by placement in operable linkage with aregulatable promoter). In a specific example, pH and/or nitrogen levelis used to regulate an amt03 promoter. The environmental condition maythen be modulated to tune the cell to produce the desired amount ofstearate appearing in cell triglycerides (e.g., to twice the oleateconcentration). As a result of these manipulations, the cell may exhibitan increase in stearate of at least 5, 10, 15, or 20 fold. In additionto stearate, arachidic, behenic, lignoceric, and cerotic acids may alsobe produced.

Alternately, the cell can be engineered to favor formation ofSatUnsatSat where Sat is palmitate or a mixture of palmitate andstearate. In this case introduction of an exogenous palmitate liberatingacyl-ACP thioesterase can promote palmitate formation. In thisembodiment, the cell can produce triglycerides, that are at least 30,40, 50, 60, 70, or 80% POP, or triglycerides in which the sum of POP,SOS, and POS is at least 30, 40, 50, 60, 70, 80, or 90% of celltriglycerides. In other related embodiments, the POS level is at least30, 40, 50, 60, 70, 80, or 90% of the triglycerides produced by thecell.

In a specific embodiment, the melting temperature of the oil is similarto that of cocoa butter (about 30-32° C.). The POP, POS and SOS levelscan approximate cocoa butter at about 16, 38, and 23% respectively. Forexample, POP can be 16%±20%, POS can be 38%±20%, an SOS can be 23%±20%.Or, POP can be 16%±15%, POS can be 38%±15%, an SOS can be 23%±15%. Or,POP can be 16%±10%, POS can be 38%±10%, an SOS can be 23%±10%.

As a result of the recombinant nucleic acids that increase stearate, aproportion of the fatty acid profile may be arachidic acid. For example,the fatty acid profile can be 0.01% to 5%, 0.1 to 4%, or 1 to 3%arachidic acid. Furthermore, the regiospecific profile may have 0.01% to4%, 0.05% to 3%, or 0.07% to 2% AOS, or may have 0.01% to 4%, 0.05% to3%, or 0.07% to 2% AOA. It is believed that AOS and AOA may reduceblooming and fat migration in confection comprising the fats of thepresent invention, among other potential benefits.

In addition to the manipulations designed to increase stearate and/orpalmitate, the levels of polyunsaturates may be suppressed, including asdescribed above by reducing SAD activity and optionally supplementingthe growth medium or regulating SAD expression. It has been discoveredthat, in microalgae (as evidenced by work in Prototheca strains),polyunsaturates are preferentially added to the sn-2 position. Thus, toelevate the percent of triglycerides with oleate at the sn-2 position,production of linoleic acid by the cell may be suppressed. Thetechniques described herein, in connection with highly oxidativelystable oils, for inhibiting or ablating fatty acid desaturase (FAD)genes or gene products may be applied with good effect toward producingSatUnsatSat oils by reducing polyunsaturates at the sn-2 position. As anadded benefit, such oils can have improved oxidatively stability. Asalso described herein, the fats may be produced in two stages withpolyunsaturates supplied or produced by the cell in the first stage witha deficit of polyunsaturates during the fat producing stage. The fatproduced may have a fatty acid profile having less than or equal to 15,10, 7, 5, 4, 3, 2, 1, or 0.5% polyunsaturates.

In an embodiment, the natural fat is a shea stearin substitute having65% to 95% SOS and optionally 0.001 to 5% SSS. In a related embodiment,the fat has 65% to 95% SOS, 0.001 to 5% SSS, and optionally 0.1 to 8%arachidic acid containing triglycerides. In another related embodiment,the fat has 65% to 95% SOS and the sum of SSS and SSO is less than 10%or less than 5%.

The cell's regiospecific preference can be learned using the analyticalmethod described below (Examples 1-2, 8). Despite balancing thesaturates and unsaturates as describe above, it is possible that thecell enzymes do not place the unsaturated fatty acid at the sn-2position. In this case, genetic manipulations can confer the desiredregiospecificity by (i) reducing the activity of endogenous sn-2specific acyl transferases (e.g., LPAAT) and/or (ii) introducing anexogenous LPAAT with the desired specificity (i.e., introduction ofoleate at sn-2). Where an exogenous LPAAT is introduced, preferably thegene encoding the LPAAT is integrated into a host chromosome and istargeted to the endoplasmic reticulum. In some cases, the host cell mayhave both specific and non-specific LPAAT alleles and suppressing theactivity of one of these alleles (e.g., with a gene knockout) willconfer the desired specificity.

In an embodiment, fats produced by cells according to the invention areused to produce a confection, candy coating, or other food product. As aresult, a food product like a chocolate or candy bar may have the “snap”(e.g., when broken) of a similar product produced using cocoa butter.The fat used may be in a beta polymorphic form or tend to a betapolymorphic form. In an embodiment, a method includes adding such a fatto a confection. Optionally, the fat can be a cocoa butter equivalentper EEC regulations, having greater than 65% SOS, less than 45%unsaturated fatty acid, less than 5% unsaturated fatty acids, less than1% lauric acid, and less than 2% trans fatty acid. The fats can also beused as cocoa butter extenders, improvers, replacers, or anti-bloomingagents, or as shea butter replacers, including in food and personal careproducts. High SOS fats produced using the cells and methods disclosedhere can be used in any application or formulation that calls for sheabutter or shea fraction. However, unlike shea butter, fats produced bythe embodiments of the invention can have low amounts ofunsaponifiables; e.g. less than 7, 5, 3, or 2% unsaponifiables. Inaddition, shea butter tends to degrade quickly due to the presence ofdiacylglycerides whereas fats produced by the embodiments of theinvention can have low amounts of diacylglycerides; e.g., less than 5,4, 3, 2, 1, or 0.5% diacylglycerides.

In an embodiment of the invention there is a natural fat suitable as ashortening, and in particular, as a roll-in shortening. Thus, theshortening may be used to make pastries or other multi-laminate foods.The shortening can be produced using methods disclosed herein forproducing engineered organisms and especially heterotrophic microalgae.In an embodiment, the shortening has a melting temperature of between 40to 60° C. and preferably between 45-55° C. and can have a triglycerideprofile with 15 to 20% medium chain fatty acids (C8 to C14), 45-50% longchain saturated fatty acids (myristic and palmitic acid), and 30-35%unsaturated fatty acids (preferably with more oleic than linoleic). Theshortening may form β′ polymorphic crystals, optionally without passingthrough the β polymorphic form. The shortening may be thixotrophic. Theshortening may have a solid fat content of less than 15% at 35° C. In aspecific embodiment, there is a natural oil suitable as a roll-inshortening produced by a recombinant microalga, where the oil has ayield stress between 400 and 700 or 500 and 600 Pa and a storage modulusof greater than 1×10⁵ Pa or 1×10⁶ Pa. (see Example 46)

A structured solid-liquid fat system can be produced using thestructuring oils by blending them with an oil that is a liquid at roomtemperature (e.g., an oil high in tristearin or triolein). The blendedsystem may be suitable for use in a food spread, mayonnaise, dressing,shortening; i.e. by forming an oil-water-oil emulsion. The structuringfats according to the embodiments described here, and especially thosehigh in SOS, can be blended with other oils/fats to make a cocoa butterequivalent, replacer, or extender. For example, a natural fat havinggreater than 65% SOS can be blended with palm mid-fraction to make acocoa butter equivalent.

In general, such high Sat-Unsat-Sat fats or fat systems can be used in avariety of other products including whipped toppings, margarines,spreads, salad dressings, baked goods (e.g. breads, cookies, crackersmuffins, and pastries), cheeses, cream cheese, mayonnaise, etc.

In a specific embodiment, a Sat-Unsat-Sat fat described above is used toproduce a margarine, spread, or the like. For example, a margarine canbe made from the fat using any of the recipes or methods found in U.S.Pat. Nos. 7,118,773, 6,171,636, 4447462, 5690985, 5888575, 5972412,6171636, or international patent publications WO9108677A1.

In an embodiment, a fat comprises a natural fat optionally blended withanother fat and is useful for producing a spread or margarine or otherfood product is produced by the genetically engineered cell and hasglycerides derived from fatty acids which comprises:

-   -   (a) at least 10 weight % of C18 to C24 saturated fatty acids,    -   (b) which comprise stearic and/or arachidic and/or behenic        and/or lignoceric acid and    -   (c) oleic and/or linoleic acid, while    -   (d) the ratio of saturated C18 acid/saturated        (C20+C22+C24)-acids≧1, preferably ≧5, more preferably ≧10, which        glycerides contain:    -   (e) ≦5 weight % of linolenic acid calculated on total fatty acid        weight    -   (f) ≦5 weight % of trans fatty acids calculated on total fatty        acid weight    -   (g) ≦75 weight %, preferably ≦60 weight % of oleic acid at the        sn-2 position: which glycerides contain calculated on total        glycerides weight    -   (h) ≧8 weight % HOH+HHO triglycerides    -   (i) ≦5 weight % of trisaturated triglycerides, and optionally        one or more of the following properties:    -   (j) a solid fat content of >10% at 10° C.    -   (k) a solid fat content≦15% at 35° C.,    -   (l) a solid fat content of >15% at 10° C. and a solid fat        content≦25% at 35° C.,    -   (m) the ratio of (HOH+HHO) and (HLH+HHL) triglycerides is >1,        and preferably >2,        -   where H stands for C18-C24 saturated fatty acid, O for oleic            acid, and L for linoleic acid.

Optionally, the solid content of the fat (% SFC) is 11 to 30 at 10° C.,4 to 15 at 20° C., 0.5 to 8 at 30° C., and 0 to 4 at 35° C. Alternately,the % SFC of the fat is 20 to 45 at 10° C., 14 to 25 at 20° C., 2 to 12at 30° C., and 0 to 5 at 35° C. In related embodiment, the % SFC of thefat is 30 to 60 at 10° C., 20 to 55 at 20° C., 5 to 35 at 30° C., and 0to 15 at 35° C. The C12-C16 fatty acid content can be ≦15 weight %. Thefat can have ≦5 weight % disaturated diglycerides.

In related embodiments there is a spread, margarine or other foodproduct made with the natural oil or natural oil blend. For example, thenatural fat can be used to make an edible W/O emulsion spread comprising70-20 wt. % of an aqueous phase dispersed in 30-80 wt. % of a fat phasewhich fat phase is a mixture of 50-99 wt. % of a vegetable triglycerideoil A and 1-50 wt. % of a structuring triglyceride fat B, which fatconsists of 5-100 wt. % of a hardstock fat C and up to 95 wt. % of a fatD, where at least 45 wt. % of the hardstock fat C triglycerides consistof SatOSat triglycerides and where Sat denotes a fatty acid residue witha saturated C18-C24 carbon chain and O denotes an oleic acid residue andwith the proviso that any hardstock fat C which has been obtained byfractionation, hydrogenation, esterification or interesterification ofthe fat is excluded. The hardstock fat can be a natural fat produced bya cell according to the methods disclosed herein. Accordingly, thehardstock fat can be a fat having a regiospecific profile having atleast 50, 60, 70, 80, or 90% SOS. The W/0 emulsion can be prepared tomethods known in the art including in U.S. Pat. No. 7,118,773.

In related embodiment, the cell also expresses an endogenous hydrolyaseenzyme that produces ricinoleic acid. As a result, the oil (e.g., aliquid oil or structured fat) produced may be more easily emulsifiedinto a margarine, spread, or other food product or non-food product. Forexample, the oil produced may be emulsified using no added emulsifiersor using lower amounts of such emulsifiers. The U.S. patent applicationSer. No. 13/365,253 discloses methods for expressing such hydroxylasesin microalgae and other cells. In specific embodiments, a natural oilcomprises at least 1, 2, or 5% SRS, where S is stearate and R isricinoleic acid.

In an alternate embodiment, a natural oil that is a cocoa butter mimeticas described above can be fractionated to remove trisaturates (e.g.,tristearin and tripalmitin, SSP, and PPS). For example, it has beenfound that microalgae engineered to decrease SAD activity to increaseSOS concentration make an oil that can be fractionated to removetrisaturated. See Example 47. In specific embodiments, the meltingtemperature of the fractionated natural oil is similar to that of cocoabutter (about 30-32° C.). The POP, POS and SOS levels can approximatecocoa butter at about 16, 38, and 23% respectively. For example, POP canbe 16%±20%, POS can be 38%±20%, an SOS can be 23%±20%. Or, POP can be16%±15%, POS can be 38%±15%, an SOS can be 23%±15%. Or, POP can be16%±10%, POS can be 38%±10%, an SOS can be 23%±10%. In addition, thetristearin levels can be less than 5% of the triacylglycerides.

VIII. High Mid-Chain Oils

In an embodiment of the present invention, the cell has recombinantnucleic acids operable to elevate the level of midchain fatty acids inthe cell or in the oil of the cell. One way to increase the levels ofmidchain fatty acids in the cell or in the oil of the cell is toengineer a cell to express an exogenous acyl-ACP thioesterase that hasactivity towards midchain fatty acyl-ACP substrates, either as a solemodification or in combination with one or more other geneticmodifications. An additional genetic modification to increase the levelof midchain fatty acids in the cell or oil of the cell is the expressionof an exogenous lysophosphatidic acid acyltransferase gene encoding anactive lysophosphatidic acid acyltransferase (LPAAT) that catalyzes thetransfer of a mid-chain fatty-acyl group to the sn-2 position of asubstituted acylglyceroester. In a specific related embodiment, both anexogenous acyl-ACP thioesterase and LPAAT are stably expressed in thecell. As a result of introducing recombinant nucleic acids into anoleaginous cell (and especially into a plastidic microbial cell) anexogenous mid-chain-specific thioesterase and an exogenous LPAAT thatcatalyzes the transfer of a mid-chain fatty-acyl group to the sn-2position of a substituted acylglyceroester, the cell can be made toincrease the percent of fatty acid in the TAGs that it produces by 10,20 30, 40, 50, 60, 70, 80, 90-fold, or more. Introduction of theexogenous LPAAT can increase midchain fatty acids at the sn-2 positionby 1.2, 1, 5, 1.7, 2, 3 4 fold or more compared to introducing anexogenous mid-chain preferring acyl-ACP thioesterase alone. In anembodiment, the mid-chain fatty acid is greater than 30, 40, 50 60, 70,80, or 90% of the TAG fatty acids produced by the cell. In variousembodiments, the mid-chain fatty acid is lauric, myristic, or palmitic.Examples 3, 43, and 44 describe expression of plant LPAATs in microalgalcells with resulting alterations in fatty acid profiles. As in theexamples, the cells can also express an exogenous acyl-ACP thioesterase(which can also be from a plant) with a preference for a given fattyacyl-ACP chain length. For example, a microalgal cell can compriseexogenous genes encoding a LPAAT and an acyl-ACP thioesterase thatpreferentially cleave C8, C10, C12, C14, C8-C12, or C8-C10 fatty acids.In a specific embodiment, such a cell is capable of producing a naturaloil with a fatty acid profile comprising 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, or90-99%, >20%, >30%, >40%, >50%, >60%, >70%, >80% or >90% C8, C10, C12,C14, C8-C12, or C8-C10 fatty acids. Other LPAAT can preferentiallycleave C16 or C18 fatty acids (see Example 44). Further geneticmanipulation of the fatty acid desaturase pathway (e.g., as describedinfra) can increase the stability of the oils. Any of these natural oilscan be interesterified. Interesterification can, for example, be used tolower the melting temperature or pour-point of the oil. In a specificembodiment, the natural oil comprises at least 50% of the sum ofcaprylic and capric acids and may be interesterified to reduce the pourpoint and/or kinematic viscosity. Such an oil (natural orinteresterified) can optionally be a high stability oil comprising, forexample, less than 2% polyunsaturated fatty acids.

Alternately, or in addition to expression of an exogenous LPAAT, thecell may comprise recombinant nucleic acids that are operable to expressan exogenous KASI or KASIV enzyme and optionally to decrease oreliminate the activity of a KASII, which is particularly advantageouswhen a mid-chain-preferring acyl-ACP thioesterase is expressed. Example37 describes the engineering of Prototheca cells to overexpress KASI orKASIV enzymes in conjunction with a mid-chain preferring acyl-ACPthioesterase to generate strains in which production of C10-C12 fattyacids is about 59% of total fatty acids. Mid-chain production can alsobe increased by suppressing the activity of KASI and/or KASII (e.g.,using a knockout or knockdown). Example 38 details the chromosomalknockout of different alleles of Prototheca moriformis (UTEX 1435) KASIin conjunction with overexpression of a mid-chain preferring acyl-ACPthioesterase to achieve fatty acid profiles that are about 76% or 84%C10-C14 fatty acids. Example 39 provides recombinant cells and oilscharacterized by elevated midchain fatty acids as a result of expressionof KASI RNA hairpin polynucleotides. In addition to any of thesemodifications, unsaturated or polyunsaturated fatty acid production canbe suppressed (e.g., by knockout or knockdown) of a SAD or FAD enzyme.

In a particular embodiment, a recombinant cell produces TAG having 40%lauric acid or more. In another related embodiment, a recombinant cellproduces TAG having a fatty acid profile of 40% or more of myristic,caprylic, capric, or palmitic acid. For example, an oleaginousrecombinant clorophyte cell can produce 40% lauric or myristic acid inan oil that makes up 40, 50, or 60% or more of the cell's dry weight.

In a specific embodiment, a recombinant cell comprises nucleic acidsoperable to express a product of an exogenous gene encoding alysophosphatidic acid acyltransferase that catalyzes the transfer of amid-chain fatty-acyl group to the sn-2 position of a substitutedacylglyceroester and nucleic acids operable to express a product of anacyl-ACP thioesterase exogenous gene encoding an active acyl-ACPthioesterase that catalyzes the cleavage of mid-chain fatty acids fromACP. As a result, in one embodiment, the oil produced can becharacterized by a fatty acid profile elevated in C10 and C12 fattyacids and reduced in C16, C18, and C18:1 fatty acids as a result of therecombinant nucleic acids. See Example 3, in which overexpression of aCuphea wrightii acyl-ACP thioesterase and a Cocos nucifera LPAAT geneincreased the percentage of C12 fatty acids from about 0.04% in theuntransformed cells to about 46% and increased the percentage of C10fatty acids from about 0.01% in the untransformed cells to about 11%. Inrelated embodiments, the increase is greater than 70%, from 75-85%, from70-90%, from 90-200%, from 200-300%, from 300-400%, from 400-500%, orgreater than 500%.

Average chain length can also be reduced by overexpression of aC18-specific acyl-ACP thioesterase. Recombinant nucleic acids operableto overexpress a C18 or other acyl-ACP thioesterase may be used alone orin combination with the other constructs described here to furtherreduce average chain length. Among other uses, the oils produced can beused as cocoa-butter/milkfat substitute. See Example 45 and thediscussion of FIG. 17. In an embodiment, one of the above described highmid-chain producing cells is further engineered to produce a lowpolyunsaturated oil by knocking out or knocking down one or more fattyacyl desturases, as described above in section IV. Accordingly, the oilproduced can have the high stability characteristic mentioned in thatsection or in corresponding Examples.

The high mid-chain oils or fatty acids derived from hydrolysis of theseoils may be particularly useful in food, fuel and oleochemicalapplications including the production of lubricants and surfactants. Forexample, fatty acids derived from the cells can be esterified, cracked,reduced to an aldehyde or alcohol, aminated, sulfated, sulfonated, orsubjected to other chemical process known in the art.

In some embodiments, the natural oil is interesterified and thekinematic viscosity of the interesterified natural oil is less than 30,20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 centiStokes at 40° C. In someembodiments, the kinematic viscosity is less than 3 centiStokes at 40°C. In some embodiments, the pour point of an interesterified natural oilis less than, 5° C., 0° C., −10° C., −12° C., −15° C., −20° C., −25° C.,−30° C., −35° C., −40° C., −45° C., or −50° C. In some embodiments, thepour point is less than −10° C. In some embodiments, the pour point isless than −20° C.

IX. High Oleic/Palmitic Oil

In another embodiment, there is a high oleic oil with about 60% oleicacid, 25% palmitic acid and optionally 5% polyunsaturates or less. Thehigh oleic oil can be produced using the methods disclosed in U.S.patent application Ser. No. 13/365,253, which is incorporated byreference in relevant part. For example, the cell can have nucleic acidsoperable to suppress an acyl-ACP thioesterase (e.g., knockout orknockdown of a gene encoding FATA) while also expressing an gene thatincreases KASII activity.

X. Low Saturate Oil

In an embodiment, a natural oil is produced from a recombinant cell. Theoil produced has a fatty acid profile that has less that 4%, 3%, 2%, 1%,or saturated fatty acids. In a specific embodiment, the oil has 0.1 to3.5% saturated fat. Certain of such oils can be used to produce a foodwith negligible amounts of saturated fatty acids. Optionally, these oilscan have fatty acid profiles comprising at least 90% oleic acid or atleast 90% oleic acid with at least 3% polyunsaturated fatty acids. In anembodiment, a natural oil produced by a recombinant cell comprises atleast 90% oleic acid, at least 3% of the sum of linoleic and linolenicacid and has less than 3.5% saturated fatty acids. In a relatedembodiment, a natural oil produced by a recombinant cell comprises atleast 90% oleic acid, at least 3% of the sum of linoleic and linolenicacid and has less than 3.5% saturated fatty acids, the majority of thesaturated fatty acids being comprised of chain length 10 to 16. Theseoils may be produced by recombinant oleaginous cells including but notlimited to those described here and in U.S. patent application Ser. No.13/365,253. For example, overexpression of a KASII enzyme in a cell witha highly active SAD can produce a high oleic oil with less than or equalto 3.5% saturates. Optionally, an oleate-specific acyl-ACP thioesteraseis also overexpressed and/or an endogenous thioesterase having apropensity to hydrolyze acyl chains of less than C18 knocked out orsuppressed. The oleate-specific acyl-ACP thioesterase may be a transgenewith low activity toward ACP-palmitate and ACP-stearate so that theratio of oleic acid relative to the sum of palmitic acid and stearicacid in the fatty acid profile of the oil produced is greater than 3, 5,7, or 10. Alternately, or in addition, a FATA gene may be knocked out orknocked down, as in Example 36 below. Another optional modification isto increase KASI and/or KASIII activity, which can further suppress theformation of shorter chain saturates. Optionally, one or moreacyltransferases having specificity for transferring unsaturated fattyacyl moieties to a substituted glycerol is also overexpressed and/or anendogenous acyltransferase is knocked out or attenuated. An additionaloptional modification is to increase the activity of KCS enzymes havingspecificity for elongating unsaturated fatty acids and/or an endogenousKCS having specificity for elongating saturated fatty acids is knockedout or attenuated.

As described in Example 51, levels of saturated fats may reduced byintroduction of an exogenous gene that desaturates palmitic acid topalmitoleic acid. Examples of suitable genes for use in the oleaginouscells are found in the plants, including Macfadyena unguis (Cat's claw),Macadamia integrifolia (Macadamia nut) and Hippophae rhamnoides (seabuckthorn). Variant exogenous or endogenous SADs that desaturatepalmitoyl-ACP can also be used and are further discussed in Example 51.This modification can be used alone, or in combination witholeate-increasing modifications such as those described in section IXand the Examples, including knockout or knockdown of one or moreendogenous FATA alleles. In one embodiment, an oleaginous cell such asan oleaginous microalgae has a combination of (i) a FATA knockout orknockdown with (ii) expression of an exogenous PAD gene (this could alsobe a variant SAD with PAD activity) and/or a mutation in an endogenousSAD gene to give PAD activity. Such as cell may further comprise anoverexpressed endogenous or exogenous KASII gene. In accordance with anyof these embodiments of the invention, the oleaginous cell produces anoil having a fatty acid profile with 1-2, 2-3, 3-4, 5-6, 7-8, 9-10,10-15, 15-20, 20-30, 30-40, 40-60, 60-70, 70-80, 80-90, or 90-100 areapercent palmitoleic acid. In a specific embodiment, the cell producesgreater than 50% oleic acid, greater than 1% palmitoleic acid, an 3.5area % or less of saturated fatty acids.

In addition to the above genetic modifications, the low saturate oil canbe a high-stability oil by virtue of low amounts of polyunsaturatedfatty acids. Methods and characterizations of high-stability,low-polyunsaturated oils are described in the section above entitled LowPolyunsaturated Oils, including method to reduce the activity ofendogenous Δ12 fatty acid desaturase. In a specific embodiment, an oilis produced by a oleaginous microbial cell having a type II fatty acidsynthetic pathway and has no more than 3.5% saturated fatty acids andalso has no more than 3% polyunsaturated fatty acids. In anotherspecific embodiment, the oil has no more than 3% saturated fatty acidsand also has no more than 2% polyunsaturated fatty acids. In anotherspecific embodiment, the oil has no more than 3% saturated fatty acidsand also has no more than 1% polyunsaturated fatty acids.

The low saturate and low saturate/high stability oil can be blended withless expensive oils to reach a targeted saturated fatty acid level atless expense. For example, an oil with 1% saturated fat can be blendedwith an oil having 7% saturated fat (e.g. high-oleic sunflower oil) togive an oil having 3% saturated fat.

Oils produced according to embodiments of the present invention can beused in the transportation fuel, oleochemical, and/or food and cosmeticindustries, among other applications. For example, transesterificationof lipids can yield long-chain fatty acid esters useful as biodiesel.Other enzymatic and chemical processes can be tailored to yield fattyacids, aldehydes, alcohols, alkanes, and alkenes. In some applications,renewable diesel, jet fuel, or other hydrocarbon compounds are produced.The present disclosure also provides methods of cultivating microalgaefor increased productivity and increased lipid yield, and/or for morecost-effective production of the compositions described herein. Themethods described here allow for the production of oils from plastidiccell cultures at large scale; e.g., 1000, 10,000, 100,000 liters ormore.

XI. Cocoa Butter/Milk-Fat Blend Mimetics

In certain embodiments, the cell produces a natural oil that has atemperature-dependent solid fat content (“SFC-curve”) that approximatesa blend of cocoa butter and milkfat. Such oils may be used where thecocoa butter/milkfat blend could be used; for example, in chocolatesother confections, ice cream or other frozen desserts, pastries, ordough, including for quickbreads, or other baked goods. The oils mayinhibit blooming, enhance flavor, enhance texture, or reduce costs. In aspecific example, the natural oil approximates. Accordingly, anembodiment of the invention is using a natural oil from a recombinantmicroalgal cell to replace a cocoa butter/milkfat blend in a recipe. Ina related embodiment,

FIG. 17 shows a plot of % solid fat content for various oils as follows(a) P. moriformis RBD oil without lipid pathway engineering, (b)Brazilian cocoa butter+25% milkfat, (c) three replicates of P.moriformis RBD oil from a strain expressing hairpin nucleic acids thatreduce levels of a SAD allele thus reducing oleic acid and increasingstearic acid in the TAG profile, (d) P. moriformis RBD oil from a strainoverexpressing an endogenous OTE (oleoyl acyl-ACP thioesterase, seeExample 45), (e) Malaysian cocoa butter+25% milkfat, and (f) Malaysiancocoa butter. The cocoa butter and cocoa butter milkfat values areliterature values (Bailey's Industrial Oils and Fat Products, 6^(th)ed.)

In an embodiment of the present invention, a natural oil that is similarin thermal properties to a 75% cocoa butter/25% milkfat blend isproduced by a microalgal or other cell described above. The cellcomprises recombinant nucleic acids operable to alter the fatty acidprofile of triglycerides produced by the cell so as that the oil has asolid fat content (e.g., as determined by NMR) of 38%±30% at 20° C.,32%±30% at 25° C., 17%±30% at 30° C., and less than 5%±30% at 35° C. Forthe sake of clarity, ±10% refers to percent of the percent SFC (e.g.,30% of 5% SFC is 1.5% SFC so the range is 3.5 to 6.5% SFC at 35° C.). Inrelated embodiments, the oil has a solid fat content (e.g., asdetermined by NMR) of 38%±20% at 20° C., 32%±20% at 25° C., 17%±20% at30° C., and less than 5%±20% at 35° C. or the oil has a solid fatcontent (e.g., as determined by NMR) of 38%±10% at 20° C., 32%±10% at25° C., 17%±10% at 30° C., and less than 5%±10% at 35° C.

XII. Minor Oil Components

The oils produced according to the above methods in some cases are madeusing a microalgal host cell. As described above, the microalga can be,without limitation, fall in the classification of Chlorophyta,Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It hasbeen found that microalgae of Trebouxiophyceae can be distinguished fromvegetable oils based on their sterol profiles. Oil produced by Chlorellaprotothecoides was found to produce sterols that appeared to bebrassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol,when detected by GC-MS. However, it is believed that all sterolsproduced by Chlorella have C24β stereochemistry. Thus, it is believedthat the molecules detected as campesterol, stigmasterol, andβ-sitosterol, are actually 22,23-dihydrobrassicasterol, proferasteroland clionasterol, respectively. Thus, the oils produced by themicroalgae described above can be distinguished from plant oils by thepresence of sterols with C24β stereochemistry and the absence of C24αstereochemistry in the sterols present. For example, the oils producedmay contain 22,23-dihydrobrassicasterol while lacking campesterol;contain clionasterol, while lacking in β-sitosterol, and/or containporiferasterol while lacking stigmasterol. Alternately, or in addition,the oils may contain significant amounts of Δ⁷-poriferasterol.

XIII. Fuels and Chemicals

The oils discussed above alone or in combination are useful in theproduction of foods, fuels and chemicals (including plastics, foams,films, etc). The oils, triglycerides, fatty acids from the oils may besubjected to C—H activation, hydroamino methylation,methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation,methylation, dimerization, thiolation, metathesis, hydro-alkylation,lactonization, or other chemical processes.

The oils can be converted to alkanes (e.g., renewable diesel) or esters(e.g., methyl or ethyl esters for biodisesel produced bytransesterification). The alkanes or esters may be used as fuel, assolvents or lubricants, or as a chemical feedstock. Methods forproduction of renewable diesel and biodiesel are well established in theart. See, for example, WO2011/150411.

In a specific embodiment of the present invention, a high-oleic orhigh-oleic-high stability oil described above is esterified. Forexample, the oils can be transesterified with methanol to an oil that isrich in methyl oleate. As described in Example 49, such formulationshave been found to compare favorably with methyl oleate from soybeanoil.

In another specific example, the oil is converted to C36 diacids orproducts of C36 diacids. Fatty acids produced from the oil can bepolymerized to give a composition rich in C36 dimer acids. In a specificexample, high-oleic oil is split to give a high-oleic fatty acidmaterial which is polymerized to give a composition rich in C36-dimeracids. It is believed that using a high oleic starting material willgive lower amounts of cyclic products, which may be desirable in somecases. Further, the C36 dimer acids can be esterified and hydrogenatedto give diols. The diols can be polymerized by catalytic dehydration.Polymers can also produced by tranesterification of dimerdiols withdimethyl carbonate.

For the production of fuel in accordance with the methods of theinvention lipids produced by cells of the invention are harvested, orotherwise collected, by any convenient means. Lipids can be isolated bywhole cell extraction. The cells are first disrupted, and thenintracellular and cell membrane/cell wall-associated lipids as well asextracellular hydrocarbons can be separated from the cell mass, such asby use of centrifugation. Intracellular lipids produced in oleaginouscells are, in some embodiments, extracted after lysing the cells. Onceextracted, the lipids are further refined to produce oils, fuels, oroleochemicals.

Various methods are available for separating lipids from cellularlysates. For example, lipids and lipid derivatives such as fattyaldehydes, fatty alcohols, and hydrocarbons such as alkanes can beextracted with a hydrophobic solvent such as hexane (see Frenz et al.1989, Enzyme Microb. Technol., 11:717). Lipids and lipid derivatives canalso be extracted using liquefaction (see for example Sawayama et al.1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, BiomassBioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al.1995, Fuel 74(12):1735-1738); and supercritical CO₂ extraction (see forexample Mendes et al. 2003, Inorganica Chimica Acta 356:328-334). Miaoand Wu describe a protocol of the recovery of microalgal lipid from aculture of Chlorella prototheocoides in which the cells were harvestedby centrifugation, washed with distilled water and dried by freezedrying. The resulting cell powder was pulverized in a mortar and thenextracted with n-hexane. Miao and Wu, Biosource Technology (2006)97:841-846.

Lipids and lipid derivatives can be recovered by extraction with anorganic solvent. In some cases, the preferred organic solvent is hexane.Typically, the organic solvent is added directly to the lysate withoutprior separation of the lysate components. In one embodiment, the lysategenerated by one or more of the methods described above is contactedwith an organic solvent for a period of time sufficient to allow thelipid and/or hydrocarbon components to form a solution with the organicsolvent. In some cases, the solution can then be further refined torecover specific desired lipid or hydrocarbon components. Hexaneextraction methods are well known in the art.

Lipids produced by cells in vivo, or enzymatically modified in vitro, asdescribed herein can be optionally further processed by conventionalmeans. The processing can include “cracking” to reduce the size, andthus increase the hydrogen:carbon ratio, of hydrocarbon molecules.Catalytic and thermal cracking methods are routinely used in hydrocarbonand triglyceride oil processing. Catalytic methods involve the use of acatalyst, such as a solid acid catalyst. The catalyst can besilica-alumina or a zeolite, which result in the heterolytic, orasymmetric, breakage of a carbon-carbon bond to result in a carbocationand a hydride anion. These reactive intermediates then undergo eitherrearrangement or hydride transfer with another hydrocarbon. Thereactions can thus regenerate the intermediates to result in aself-propagating chain mechanism. Hydrocarbons can also be processed toreduce, optionally to zero, the number of carbon-carbon double, ortriple, bonds therein. Hydrocarbons can also be processed to remove oreliminate a ring or cyclic structure therein. Hydrocarbons can also beprocessed to increase the hydrogen:carbon ratio. This can include theaddition of hydrogen (“hydrogenation”) and/or the “cracking” ofhydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described below inthis Section.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manufacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is an urgent need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass or otherwise that meets the appropriateASTM specification can be defined as diesel fuel (ASTM D975), jet fuel(ASTM D1655), or as biodiesel if it is a fatty acid methyl ester (ASTMD6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear. Typically, biodiesel comprises C14-C18 alkyl esters.Various processes convert biomass or a lipid produced and isolated asdescribed herein to diesel fuels. A preferred method to producebiodiesel is by transesterification of a lipid as described herein. Apreferred alkyl ester for use as biodiesel is a methyl ester or ethylester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B100.

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel. The lipid profile of the biodiesel isusually highly similar to the lipid profile of the feedstock oil.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases. In a base-catalyzed transesterification process, thetriacylglycerides are reacted with an alcohol, such as methanol orethanol, in the presence of an alkaline catalyst, typically potassiumhydroxide. This reaction forms methyl or ethyl esters and glycerin(glycerol) as a byproduct.

Transesterification has also been carried out, as discussed above, usingan enzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80° C., and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1. Lipasessuitable for use in transesterification include, but are not limited to,those listed in Table 9. Other examples of lipases useful fortransesterification are found in, e.g. U.S. Pat. Nos. 4,798,793;4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032. Such lipasesinclude, but are not limited to, lipases produced by microorganisms ofRhizopus, Aspergillus, Candida, Mucor, Pseudomonas, Rhizomucor, Candida,and Humicola and pancreas lipase.

Subsequent processes may also be used if the biodiesel will be used inparticularly cold temperatures. Such processes include winterization andfractionation. Both processes are designed to improve the cold flow andwinter performance of the fuel by lowering the cloud point (thetemperature at which the biodiesel starts to crystallize). There areseveral approaches to winterizing biodiesel. One approach is to blendthe biodiesel with petroleum diesel. Another approach is to useadditives that can lower the cloud point of biodiesel. Another approachis to remove saturated methyl esters indiscriminately by mixing inadditives and allowing for the crystallization of saturates and thenfiltering out the crystals. Fractionation selectively separates methylesters into individual components or fractions, allowing for the removalor inclusion of specific methyl esters. Fractionation methods includeurea fractionation, solvent fractionation and thermal distillation.

Another valuable fuel provided by the methods of the present inventionis renewable diesel, which comprises alkanes, such as C10:0, C12:0,C14:0, C16:0 and C18:0 and thus, are distinguishable from biodiesel.High quality renewable diesel conforms to the ASTM D975 standard. Thelipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In one embodiment, the method for producing renewable diesel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing the microorganism to produce a lysate, (c) isolatinglipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696. Some suitable methods may include mechanicalpressing and centrifuging.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet an ASTM D975standard while leaving components that are heavier than desired formeeting the D975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

In one embodiment of the method for producing renewable diesel, treatingthe lipid to produce an alkane is performed by hydrotreating of thelipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300° to 660°F., with pressure sufficient to keep the water primarily as a liquid,100 to 170 standard atmosphere (atm). Reaction times are on the order of15 to 30 minutes. After the reaction is completed, the organics areseparated from the water. Thereby a distillate suitable for diesel isproduced.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. Nos. 5,475,160(hydrogenation of triglycerides); 5,091,116 (deoxygenation,hydrogenation and gas removal); 6,391,815 (hydrogenation); and 5,888,947(isomerization).

One suitable method for the hydrogenation of triglycerides includespreparing an aqueous solution of copper, zinc, magnesium and lanthanumsalts and another solution of alkali metal or preferably, ammoniumcarbonate. The two solutions may be heated to a temperature of about 20°C. to about 85° C. and metered together into a precipitation containerat rates such that the pH in the precipitation container is maintainedbetween 5.5 and 7.5 in order to form a catalyst. Additional water may beused either initially in the precipitation container or addedconcurrently with the salt solution and precipitation solution. Theresulting precipitate may then be thoroughly washed, dried, calcined atabout 300° C. and activated in hydrogen at temperatures ranging fromabout 100° C. to about 400° C. One or more triglycerides may then becontacted and reacted with hydrogen in the presence of theabove-described catalyst in a reactor. The reactor may be a trickle bedreactor, fixed bed gas-solid reactor, packed bubble column reactor,continuously stirred tank reactor, a slurry phase reactor, or any othersuitable reactor type known in the art. The process may be carried outeither batchwise or in continuous fashion. Reaction temperatures aretypically in the range of from about 170° C. to about 250° C. whilereaction pressures are typically in the range of from about 300 psig toabout 2000 psig. Moreover, the molar ratio of hydrogen to triglyceridein the process of the present invention is typically in the range offrom about 20:1 to about 700:1. The process is typically carried out ata weight hourly space velocity (WHSV) in the range of from about 0.1hr⁻¹ to about 5 hr⁻¹. One skilled in the art will recognize that thetime period required for reaction will vary according to the temperatureused, the molar ratio of hydrogen to triglyceride, and the partialpressure of hydrogen. The products produced by the such hydrogenationprocesses include fatty alcohols, glycerol, traces of paraffins andunreacted triglycerides. These products are typically separated byconventional means such as, for example, distillation, extraction,filtration, crystallization, and the like.

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes. Solidcatalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Suitable methods for the deoxygenation of an oil includes heating an oilto a temperature in the range of from about 350° F. to about 550° F. andcontinuously contacting the heated oil with nitrogen under at leastpressure ranging from about atmospheric to above for at least about 5minutes.

Suitable methods for isomerization include using alkali isomerizationand other oil isomerization known in the art.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the triglyceride feed. The triglyceride molecule isreduced to four hydrocarbon molecules under hydroprocessing conditions:a propane molecule and three heavier hydrocarbon molecules, typically inthe C8 to C18 range.

Thus, in one embodiment, the product of one or more chemical reaction(s)performed on lipid compositions of the invention is an alkane mixturethat comprises ASTM D975 renewable diesel. Production of hydrocarbons bymicroorganisms is reviewed by Metzger et al. Appl Microbiol Biotechnol(2005) 66: 486-496 and A Look Back at the U.S. Department of Energy'sAquatic Species Program: Biodiesel from Algae, NREL/TP-580-24190, JohnSheehan, Terri Dunahay, John Benemann and Paul Roessler (1998).

The distillation properties of a diesel fuel is described in terms ofT10-T90 (temperature at 10% and 90%, respectively, volume distilled).The T10-T90 of the material produced in Example 13 was 57.9° C. Methodsof hydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other T10-T90 ranges, such as 20, 25,30, 35, 40, 45, 50, 60 and 65° C. using triglyceride oils producedaccording to the methods disclosed herein.

Methods of hydrotreating, isomerization, and other covalent modificationof oils disclosed herein, as well as methods of distillation andfractionation (such as cold filtration) disclosed herein, can beemployed to generate renewable diesel compositions with other T10values, such as T10 between 180 and 295, between 190 and 270, between210 and 250, between 225 and 245, and at least 290.

Methods of hydrotreating, isomerization, and other covalent modificationof oils disclosed herein, as well as methods of distillation andfractionation (such as cold filtration) disclosed herein can be employedto generate renewable diesel compositions with certain T90 values, suchas T90 between 280 and 380, between 290 and 360, between 300 and 350,between 310 and 340, and at least 290.

The FBP of the material produced in Example 13 was 300° C. Methods ofhydrotreating, isomerization, and other covalent modification of oilsdisclosed herein, as well as methods of distillation and fractionation(such as cold filtration) disclosed herein, can be employed to generaterenewable diesel compositions with other FBP values, such as FBP between290 and 400, between 300 and 385, between 310 and 370, between 315 and360, and at least 300.

Other oils provided by the methods and compositions of the invention canbe subjected to combinations of hydrotreating, isomerization, and othercovalent modification including oils with lipid profiles including (a)at least 1%-5%, preferably at least 4%, C8-C14; (b) at least 0.25%-1%,preferably at least 0.3%, C8; (c) at least 1%-5%, preferably at least2%, C10; (d) at least 1%-5%, preferably at least 2%, C12; and (3) atleast 20%-40%, preferably at least 30% C8-C14.

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

The present invention also provides methods to produce jet fuel. Jetfuel is clear to straw colored. The most common fuel is anunleaded/paraffin oil-based fuel classified as Aeroplane A-1, which isproduced to an internationally standardized set of specifications. Jetfuel is a mixture of a large number of different hydrocarbons, possiblyas many as a thousand or more. The range of their sizes (molecularweights or carbon numbers) is restricted by the requirements for theproduct, for example, freezing point or smoke point. Kerosone-typeAeroplane fuel (including Jet A and Jet A-1) has a carbon numberdistribution between about 8 and 16 carbon numbers. Wide-cut ornaphta-type Aeroplane fuel (including Jet B) typically has a carbonnumber distribution between about 5 and 15 carbons.

In one embodiment of the invention, a jet fuel is produced by blendingalgal fuels with existing jet fuel. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. The lipidsproduced by the method of the present invention can be converted toolefins. The process involves flowing the lipids produced through an FCCzone and collecting a product stream comprised of olefins, which isuseful as a jet fuel. The lipids produced are contacted with a crackingcatalyst at cracking conditions to provide a product stream comprisingolefins and hydrocarbons useful as jet fuel.

In one embodiment, the method for producing jet fuel comprises (a)cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced. In one embodiment of themethod for producing a jet fuel, the lipid composition can be flowedthrough a fluid catalytic cracking zone, which, in one embodiment, maycomprise contacting the lipid composition with a cracking catalyst atcracking conditions to provide a product stream comprising C₂-C₅olefins.

In certain embodiments of this method, it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C₂-C₅ olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

Suitable FCC catalysts generally comprise at least two components thatmay or may not be on the same matrix. In some embodiments, both twocomponents may be circulated throughout the entire reaction vessel. Thefirst component generally includes any of the well-known catalysts thatare used in the art of fluidized catalytic cracking, such as an activeamorphous clay-type catalyst and/or a high activity, crystallinemolecular sieve. Molecular sieve catalysts may be preferred overamorphous catalysts because of their much-improved selectivity todesired products. IN some preferred embodiments, zeolites may be used asthe molecular sieve in the FCC processes. Preferably, the first catalystcomponent comprises a large pore zeolite, such as an Y-type zeolite, anactive alumina material, a binder material, comprising either silica oralumina and an inert filler such as kaolin.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149° C. to about 316° C. (300° F.to 600° F.). The catalyst is flowed from a blending vessel to the riserwhere it contacts the lipid composition for a time of abort 2 seconds orless.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst to lipidcomposition ratio which can vary from about 5 to about 20 and preferablyfrom about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700° C. (1292° F.) to about 760° C. (1400° F.) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO). HDO means removal of oxygen bymeans of hydrogen, that is, oxygen is removed while breaking thestructure of the material. Olefinic double bonds are hydrogenated andany sulphur and nitrogen compounds are removed. Sulphur removal iscalled hydrodesulphurization (HDS). Pretreatment and purity of the rawmaterials (lipid composition or the lipids) contribute to the servicelife of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerzation reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulphur compounds, andlight hydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerzation is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step, i.e. HDO step, of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle. In the HDO step, thepressure varies between 20 and 150 bar, preferably between 50 and 100bar, and the temperature varies between 200 and 500° C., preferably inthe range of 300-400° C. In the HDO step, known hydrogenation catalystscontaining metals from Group VII and/or VIB of the Periodic System maybe used. Preferably, the hydrogenation catalysts are supported Pd, Pt,Ni, NiMo or a CoMo catalysts, the support being alumina and/or silica.Typically, NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50-400° C. and at hydrogen pressures of 1-200 bar,preferably at a temperature between 150 and 250° C. and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapour or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner. In theisomerzation step, the pressure varies in the range of 20-150 bar,preferably in the range of 20-100 bar, the temperature being between 200and 500° C., preferably between 300 and 400° C. In the isomerizationstep, isomerization catalysts known in the art may be used. Suitableisomerization catalysts contain molecular sieve and/or a metal fromGroup VII and/or a carrier. Preferably, the isomerization catalystcontains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrierite and Pt, Pdor Ni and Al₂O₃ or SiO₂. Typical isomerization catalysts are, forexample, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂. The isomerization step and the HDO step may be carriedout in the same pressure vessel or in separate pressure vessels.Optional prehydrogenation may be carried out in a separate pressurevessel or in the same pressure vessel as the HDO and isomerizationsteps.

Thus, in one embodiment, the product of one or more chemical reactionsis an alkane mixture that comprises HRJ-5. In another embodiment, theproduct of the one or more chemical reactions is an alkane mixture thatcomprises ASTM D1655 jet fuel. In some embodiments, the compositionconforming to the specification of ASTM 1655 jet fuel has a sulfurcontent that is less than 10 ppm. In other embodiments, the compositionconforming to the specification of ASTM 1655 jet fuel has a T10 value ofthe distillation curve of less than 205° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afinal boiling point (FBP) of less than 300° C. In another embodiment,the composition conforming to the specification of ASTM 1655 jet fuelhas a flash point of at least 38° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has adensity between 775 K/M³ and 840 K/M³. In yet another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has afreezing point that is below −47° C. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anet Heat of Combustion that is at least 42.8 MJ/K. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a hydrogen content that is at least 13.4 mass %. In anotherembodiment, the composition conforming to the specification of ASTM 1655jet fuel has a thermal stability, as tested by quantitative gravimetricJFTOT at 260° C., that is below 3 mm of Hg. In another embodiment, thecomposition conforming to the specification of ASTM 1655 jet fuel has anexistent gum that is below 7 mg/dl.

Thus, the present invention discloses a variety of methods in whichchemical modification of microalgal lipid is undertaken to yieldproducts useful in a variety of industrial and other applications.Examples of processes for modifying oil produced by the methodsdisclosed herein include, but are not limited to, hydrolysis of the oil,hydroprocessing of the oil, and esterification of the oil. Otherchemical modification of microalgal lipid include, without limitation,epoxidation, oxidation, hydrolysis, sulfations, sulfonation,ethoxylation, propoxylation, amidation, and saponification. Themodification of the microalgal oil produces basic oleochemicals that canbe further modified into selected derivative oleochemicals for a desiredfunction. In a manner similar to that described above with reference tofuel producing processes, these chemical modifications can also beperformed on oils generated from the microbial cultures describedherein. Examples of basic oleochemicals include, but are not limited to,soaps, fatty acids, fatty esters, fatty alcohols, fatty nitrogencompounds including fatty amides, fatty acid methyl esters, andglycerol. Examples of derivative oleochemicals include, but are notlimited to, fatty nitriles, esters, dimer acids, quats, surfactants,fatty alkanolamides, fatty alcohol sulfates, resins, emulsifiers, fattyalcohols, olefins, drilling muds, polyols, polyurethanes, polyacrylates,rubber, candles, cosmetics, metallic soaps, soaps, alpha-sulphonatedmethyl esters, fatty alcohol sulfates, fatty alcohol ethoxylates, fattyalcohol ether sulfates, imidazolines, surfactants, detergents, esters,quats, ozonolysis products, fatty amines, fatty alkanolamides, ethoxysulfates, monoglycerides, diglycerides, triglycerides (including mediumchain triglycerides), lubricants, hydraulic fluids, greases, dielectricfluids, mold release agents, metal working fluids, heat transfer fluids,other functional fluids, industrial chemicals (e.g., cleaners, textileprocessing aids, plasticizers, stabilizers, additives), surfacecoatings, paints and lacquers, electrical wiring insulation, and higheralkanes.

Hydrolysis of the fatty acid constituents from the glycerolipidsproduced by the methods of the invention yields free fatty acids thatcan be derivatized to produce other useful chemicals. Hydrolysis occursin the presence of water and a catalyst which may be either an acid or abase. The liberated free fatty acids can be derivatized to yield avariety of products, as reported in the following: U.S. Pat. Nos.5,304,664 (Highly sulfated fatty acids); 7,262,158 (Cleansingcompositions); 7,115,173 (Fabric softener compositions); 6,342,208(Emulsions for treating skin); 7,264,886 (Water repellant compositions);6,924,333 (Paint additives); 6,596,768 (Lipid-enriched ruminantfeedstock); and 6,380,410 (Surfactants for detergents and cleaners).

In some methods, the first step of chemical modification may behydroprocessing to saturate double bonds, followed by deoxygenation atelevated temperature in the presence of hydrogen and a catalyst. Inother methods, hydrogenation and deoxygenation may occur in the samereaction. In still other methods deoxygenation occurs beforehydrogenation. Isomerization may then be optionally performed, also inthe presence of hydrogen and a catalyst. Finally, gases and naphthacomponents can be removed if desired. For example, see U.S. Pat. Nos.5,475,160 (hydrogenation of triglycerides); 5,091,116 (deoxygenation,hydrogenation and gas removal); 6,391,815 (hydrogenation); and 5,888,947(isomerization).

In some embodiments of the invention, the triglyceride oils arepartially or completely deoxygenated. The deoxygenation reactions formdesired products, including, but not limited to, fatty acids, fattyalcohols, polyols, ketones, and aldehydes. In general, without beinglimited by any particular theory, the deoxygenation reactions involve acombination of various different reaction pathways, including withoutlimitation: hydrogenolysis, hydrogenation, consecutivehydrogenation-hydrogenolysis, consecutive hydrogenolysis-hydrogenation,and combined hydrogenation-hydrogenolysis reactions, resulting in atleast the partial removal of oxygen from the fatty acid or fatty acidester to produce reaction products, such as fatty alcohols, that can beeasily converted to the desired chemicals by further processing. Forexample, in one embodiment, a fatty alcohol may be converted to olefinsthrough FCC reaction or to higher alkanes through a condensationreaction.

One such chemical modification is hydrogenation, which is the additionof hydrogen to double bonds in the fatty acid constituents ofglycerolipids or of free fatty acids. The hydrogenation process permitsthe transformation of liquid oils into semi-solid or solid fats, whichmay be more suitable for specific applications.

Hydrogenation of oil produced by the methods described herein can beperformed in conjunction with one or more of the methods and/ormaterials provided herein, as reported in the following: U.S. Pat. Nos.7,288,278 (Food additives or medicaments); 5,346,724 (Lubricationproducts); 5,475,160 (Fatty alcohols); 5,091,116 (Edible oils);6,808,737 (Structural fats for margarine and spreads); 5,298,637(Reduced-calorie fat substitutes); 6,391,815 (Hydrogenation catalyst andsulfur adsorbent); 5,233,099 and 5,233,100 (Fatty alcohols); 4,584,139(Hydrogenation catalysts); 6,057,375 (Foam suppressing agents); and7,118,773 (Edible emulsion spreads).

One skilled in the art will recognize that various processes may be usedto hydrogenate carbohydrates. One suitable method includes contactingthe carbohydrate with hydrogen or hydrogen mixed with a suitable gas anda catalyst under conditions sufficient in a hydrogenation reactor toform a hydrogenated product. The hydrogenation catalyst generally caninclude Cu, Re, Ni, Fe, Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or anycombination thereof, either alone or with promoters such as W, Mo, Au,Ag, Cr, Zn, Mn, Sn, B, P, Bi, and alloys or any combination thereof.Other effective hydrogenation catalyst materials include eithersupported nickel or ruthenium modified with rhenium. In an embodiment,the hydrogenation catalyst also includes any one of the supports,depending on the desired functionality of the catalyst. Thehydrogenation catalysts may be prepared by methods known to those ofordinary skill in the art.

In some embodiments the hydrogenation catalyst includes a supportedGroup VIII metal catalyst and a metal sponge material (e.g., a spongenickel catalyst). Raney nickel provides an example of an activatedsponge nickel catalyst suitable for use in this invention. In otherembodiment, the hydrogenation reaction in the invention is performedusing a catalyst comprising a nickel-rhenium catalyst or atungsten-modified nickel catalyst. One example of a suitable catalystfor the hydrogenation reaction of the invention is a carbon-supportednickel-rhenium catalyst.

In an embodiment, a suitable Raney nickel catalyst may be prepared bytreating an alloy of approximately equal amounts by weight of nickel andaluminum with an aqueous alkali solution, e.g., containing about 25weight % of sodium hydroxide. The aluminum is selectively dissolved bythe aqueous alkali solution resulting in a sponge shaped materialcomprising mostly nickel with minor amounts of aluminum. The initialalloy includes promoter metals (i.e., molybdenum or chromium) in theamount such that about 1 to 2 weight % remains in the formed spongenickel catalyst. In another embodiment, the hydrogenation catalyst isprepared using a solution of ruthenium(III) nitrosylnitrate, ruthenium(III) chloride in water to impregnate a suitable support material. Thesolution is then dried to form a solid having a water content of lessthan about 1% by weight. The solid may then be reduced at atmosphericpressure in a hydrogen stream at 300° C. (uncalcined) or 400° C.(calcined) in a rotary ball furnace for 4 hours. After cooling andrendering the catalyst inert with nitrogen, 5% by volume of oxygen innitrogen is passed over the catalyst for 2 hours.

In certain embodiments, the catalyst described includes a catalystsupport. The catalyst support stabilizes and supports the catalyst. Thetype of catalyst support used depends on the chosen catalyst and thereaction conditions. Suitable supports for the invention include, butare not limited to, carbon, silica, silica-alumina, zirconia, titania,ceria, vanadia, nitride, boron nitride, heteropolyacids, hydroxyapatite,zinc oxide, chromia, zeolites, carbon nanotubes, carbon fullerene andany combination thereof.

The catalysts used in this invention can be prepared using conventionalmethods known to those in the art. Suitable methods may include, but arenot limited to, incipient wetting, evaporative impregnation, chemicalvapor deposition, wash-coating, magnetron sputtering techniques, and thelike.

The conditions for which to carry out the hydrogenation reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate reaction conditions. In general, thehydrogenation reaction is conducted at temperatures of 80° C. to 250°C., and preferably at 90° C. to 200° C., and most preferably at 100° C.to 150° C. In some embodiments, the hydrogenation reaction is conductedat pressures from 500 KPa to 14000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention may include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof. As used herein, theterm “external hydrogen” refers to hydrogen that does not originate fromthe biomass reaction itself, but rather is added to the system fromanother source.

In some embodiments of the invention, it is desirable to convert thestarting carbohydrate to a smaller molecule that will be more readilyconverted to desired higher hydrocarbons. One suitable method for thisconversion is through a hydrogenolysis reaction. Various processes areknown for performing hydrogenolysis of carbohydrates. One suitablemethod includes contacting a carbohydrate with hydrogen or hydrogenmixed with a suitable gas and a hydrogenolysis catalyst in ahydrogenolysis reactor under conditions sufficient to form a reactionproduct comprising smaller molecules or polyols. As used herein, theterm “smaller molecules or polyols” includes any molecule that has asmaller molecular weight, which can include a smaller number of carbonatoms or oxygen atoms than the starting carbohydrate. In an embodiment,the reaction products include smaller molecules that include polyols andalcohols. Someone of ordinary skill in the art would be able to choosethe appropriate method by which to carry out the hydrogenolysisreaction.

In some embodiments, a 5 and/or 6 carbon sugar or sugar alcohol may beconverted to propylene glycol, ethylene glycol, and glycerol using ahydrogenolysis catalyst. The hydrogenolysis catalyst may include Cr, Mo,W, Re, Mn, Cu, Cd, Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or anycombination thereof, either alone or with promoters such as Au, Ag, Cr,Zn, Mn, Sn, Bi, B, O, and alloys or any combination thereof. Thehydrogenolysis catalyst may also include a carbonaceous pyropolymercatalyst containing transition metals (e.g., chromium, molybdenum,tungsten, rhenium, manganese, copper, cadmium) or Group VIII metals(e.g., iron, cobalt, nickel, platinum, palladium, rhodium, ruthenium,iridium, and osmium). In certain embodiments, the hydrogenolysiscatalyst may include any of the above metals combined with an alkalineearth metal oxide or adhered to a catalytically active support. Incertain embodiments, the catalyst described in the hydrogenolysisreaction may include a catalyst support as described above for thehydrogenation reaction.

The conditions for which to carry out the hydrogenolysis reaction willvary based on the type of starting material and the desired products.One of ordinary skill in the art, with the benefit of this disclosure,will recognize the appropriate conditions to use to carry out thereaction. In general, they hydrogenolysis reaction is conducted attemperatures of 110° C. to 300° C., and preferably at 170° C. to 220°C., and most preferably at 200° C. to 225° C. In some embodiments, thehydrogenolysis reaction is conducted under basic conditions, preferablyat a pH of 8 to 13, and even more preferably at a pH of 10 to 12. Insome embodiments, the hydrogenolysis reaction is conducted at pressuresin a range between 60 KPa and 16500 KPa, and preferably in a rangebetween 1700 KPa and 14000 KPa, and even more preferably between 4800KPa and 11000 KPa.

The hydrogen used in the hydrogenolysis reaction of the currentinvention can include external hydrogen, recycled hydrogen, in situgenerated hydrogen, and any combination thereof.

In some embodiments, the reaction products discussed above may beconverted into higher hydrocarbons through a condensation reaction in acondensation reactor. In such embodiments, condensation of the reactionproducts occurs in the presence of a catalyst capable of forming higherhydrocarbons. While not intending to be limited by theory, it isbelieved that the production of higher hydrocarbons proceeds through astepwise addition reaction including the formation of carbon-carbon, orcarbon-oxygen bond. The resulting reaction products include any numberof compounds containing these moieties, as described in more detailbelow.

In certain embodiments, suitable condensation catalysts include an acidcatalyst, a base catalyst, or an acid/base catalyst. As used herein, theterm “acid/base catalyst” refers to a catalyst that has both an acid anda base functionality. In some embodiments the condensation catalyst caninclude, without limitation, zeolites, carbides, nitrides, zirconia,alumina, silica, aluminosilicates, phosphates, titanium oxides, zincoxides, vanadium oxides, lanthanum oxides, yttrium oxides, scandiumoxides, magnesium oxides, cerium oxides, barium oxides, calcium oxides,hydroxides, heteropolyacids, inorganic acids, acid modified resins, basemodified resins, and any combination thereof. In some embodiments, thecondensation catalyst can also include a modifier. Suitable modifiersinclude La, Y, Sc, P, B, Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, and anycombination thereof. In some embodiments, the condensation catalyst canalso include a metal. Suitable metals include Cu, Ag, Au, Pt, Ni, Fe,Co, Ru, Zn, Cd, Ga, In, Rh, Pd, Ir, Re, Mn, Cr, Mo, W, Sn, Os, alloys,and any combination thereof.

In certain embodiments, the catalyst described in the condensationreaction may include a catalyst support as described above for thehydrogenation reaction. In certain embodiments, the condensationcatalyst is self-supporting. As used herein, the term “self-supporting”means that the catalyst does not need another material to serve assupport. In other embodiments, the condensation catalyst in used inconjunction with a separate support suitable for suspending thecatalyst. In an embodiment, the condensation catalyst support is silica.

The conditions under which the condensation reaction occurs will varybased on the type of starting material and the desired products. One ofordinary skill in the art, with the benefit of this disclosure, willrecognize the appropriate conditions to use to carry out the reaction.In some embodiments, the condensation reaction is carried out at atemperature at which the thermodynamics for the proposed reaction arefavorable. The temperature for the condensation reaction will varydepending on the specific starting polyol or alcohol. In someembodiments, the temperature for the condensation reaction is in a rangefrom 80° C. to 500° C., and preferably from 125° C. to 450° C., and mostpreferably from 125° C. to 250° C. In some embodiments, the condensationreaction is conducted at pressures in a range between 0 Kpa to 9000 KPa,and preferably in a range between 0 KPa and 7000 KPa, and even morepreferably between 0 KPa and 5000 KPa.

The higher alkanes formed by the invention include, but are not limitedto, branched or straight chain alkanes that have from 4 to 30 carbonatoms, branched or straight chain alkenes that have from 4 to 30 carbonatoms, cycloalkanes that have from 5 to 30 carbon atoms, cycloalkenesthat have from 5 to 30 carbon atoms, aryls, fused aryls, alcohols, andketones. Suitable alkanes include, but are not limited to, butane,pentane, pentene, 2-methylbutane, hexane, hexene, 2-methylpentane,3-methylpentane, 2,2,-dimethylbutane, 2,3-dimethylbutane, heptane,heptene, octane, octene, 2,2,4-trimethylpentane, 2,3-dimethyl hexane,2,3,4-trimethylpentane, 2,3-dimethylpentane, nonane, nonene, decane,decene, undecane, undecene, dodecane, dodecene, tridecane, tridecene,tetradecane, tetradecene, pentadecane, pentadecene, nonyldecane,nonyldecene, eicosane, eicosene, uneicosane, uneicosene, doeicosane,doeicosene, trieicosane, trieicosene, tetraeicosane, tetraeicosene, andisomers thereof. Some of these products may be suitable for use asfuels.

In some embodiments, the cycloalkanes and the cycloalkenes areunsubstituted. In other embodiments, the cycloalkanes and cycloalkenesare mono-substituted. In still other embodiments, the cycloalkanes andcycloalkenes are multi-substituted. In the embodiments comprising thesubstituted cycloalkanes and cycloalkenes, the substituted groupincludes, without limitation, a branched or straight chain alkyl having1 to 12 carbon atoms, a branched or straight chain alkylene having 1 to12 carbon atoms, a phenyl, and any combination thereof. Suitablecycloalkanes and cycloalkenes include, but are not limited to,cyclopentane, cyclopentene, cyclohexane, cyclohexene,methyl-cyclopentane, methyl-cyclopentene, ethyl-cyclopentane,ethyl-cyclopentene, ethyl-cyclohexane, ethyl-cyclohexene, isomers andany combination thereof.

In some embodiments, the aryls formed are unsubstituted. In anotherembodiment, the aryls formed are mono-substituted. In the embodimentscomprising the substituted aryls, the substituted group includes,without limitation, a branched or straight chain alkyl having 1 to 12carbon atoms, a branched or straight chain alkylene having 1 to 12carbon atoms, a phenyl, and any combination thereof. Suitable aryls forthe invention include, but are not limited to, benzene, toluene, xylene,ethyl benzene, para xylene, meta xylene, and any combination thereof.

The alcohols produced in the invention have from 4 to 30 carbon atoms.In some embodiments, the alcohols are cyclic. In other embodiments, thealcohols are branched. In another embodiment, the alcohols are straightchained. Suitable alcohols for the invention include, but are notlimited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol,decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol,hexadecanol, heptyldecanol, octyldecanol, nonyldecanol, eicosanol,uneicosanol, doeicosanol, trieicosanol, tetraeicosanol, and isomersthereof.

The ketones produced in the invention have from 4 to 30 carbon atoms. Inan embodiment, the ketones are cyclic. In another embodiment, theketones are branched. In another embodiment, the ketones are straightchained. Suitable ketones for the invention include, but are not limitedto, butanone, pentanone, hexanone, heptanone, octanone, nonanone,decanone, undecanone, dodecanone, tridecanone, tetradecanone,pentadecanone, hexadecanone, heptyldecanone, octyldecanone,nonyldecanone, eicosanone, uneicosanone, doeicosanone, trieicosanone,tetraeicosanone, and isomers thereof.

Another such chemical modification is interesterification. Naturallyproduced glycerolipids do not have a uniform distribution of fatty acidconstituents. In the context of oils, interesterification refers to theexchange of acyl radicals between two esters of different glycerolipids.The interesterification process provides a mechanism by which the fattyacid constituents of a mixture of glycerolipids can be rearranged tomodify the distribution pattern. Interesterification is a well-knownchemical process, and generally comprises heating (to about 200° C.) amixture of oils for a period (e.g, 30 minutes) in the presence of acatalyst, such as an alkali metal or alkali metal alkylate (e.g., sodiummethoxide). This process can be used to randomize the distributionpattern of the fatty acid constituents of an oil mixture, or can bedirected to produce a desired distribution pattern. This method ofchemical modification of lipids can be performed on materials providedherein, such as microbial biomass with a percentage of dry cell weightas lipid at least 20%.

Directed interesterification, in which a specific distribution patternof fatty acids is sought, can be performed by maintaining the oilmixture at a temperature below the melting point of some TAGs whichmight occur. This results in selective crystallization of these TAGs,which effectively removes them from the reaction mixture as theycrystallize. The process can be continued until most of the fatty acidsin the oil have precipitated, for example. A directedinteresterification process can be used, for example, to produce aproduct with a lower calorie content via the substitution oflonger-chain fatty acids with shorter-chain counterparts. Directedinteresterification can also be used to produce a product with a mixtureof fats that can provide desired melting characteristics and structuralfeatures sought in food additives or products (e.g., margarine) withoutresorting to hydrogenation, which can produce unwanted trans isomers.

Interesterification of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. Nos. 6,080,853 (Nondigestible fat substitutes); 4,288,378 (Peanutbutter stabilizer); 5,391,383 (Edible spray oil); 6,022,577 (Edible fatsfor food products); 5,434,278 (Edible fats for food products); 5,268,192(Low calorie nut products); 5,258,197 (Reduce calorie ediblecompositions); 4,335,156 (Edible fat product); 7,288,278 (Food additivesor medicaments); 7,115,760 (Fractionation process); 6,808,737(Structural fats); 5,888,947 (Engine lubricants); 5,686,131 (Edible oilmixtures); and 4,603,188 (Curable urethane compositions).

In one embodiment in accordance with the invention, transesterificationof the oil, as described above, is followed by reaction of thetransesterified product with polyol, as reported in U.S. Pat. No.6,465,642, to produce polyol fatty acid polyesters. Such anesterification and separation process may comprise the steps as follows:reacting a lower alkyl ester with polyol in the presence of soap;removing residual soap from the product mixture; water-washing anddrying the product mixture to remove impurities; bleaching the productmixture for refinement; separating at least a portion of the unreactedlower alkyl ester from the polyol fatty acid polyester in the productmixture; and recycling the separated unreacted lower alkyl ester.

Transesterification can also be performed on microbial biomass withshort chain fatty acid esters, as reported in U.S. Pat. No. 6,278,006.In general, transesterification may be performed by adding a short chainfatty acid ester to an oil in the presence of a suitable catalyst andheating the mixture. In some embodiments, the oil comprises about 5% toabout 90% of the reaction mixture by weight. In some embodiments, theshort chain fatty acid esters can be about 10% to about 50% of thereaction mixture by weight. Non-limiting examples of catalysts includebase catalysts, sodium methoxide, acid catalysts including inorganicacids such as sulfuric acid and acidified clays, organic acids such asmethane sulfonic acid, benzenesulfonic acid, and toluenesulfonic acid,and acidic resins such as Amberlyst 15. Metals such as sodium andmagnesium, and metal hydrides also are useful catalysts.

Another such chemical modification is hydroxylation, which involves theaddition of water to a double bond resulting in saturation and theincorporation of a hydroxyl moiety. The hydroxylation process provides amechanism for converting one or more fatty acid constituents of aglycerolipid to a hydroxy fatty acid. Hydroxylation can be performed,for example, via the method reported in U.S. Pat. No. 5,576,027.Hydroxylated fatty acids, including castor oil and its derivatives, areuseful as components in several industrial applications, including foodadditives, surfactants, pigment wetting agents, defoaming agents, waterproofing additives, plasticizing agents, cosmetic emulsifying and/ordeodorant agents, as well as in electronics, pharmaceuticals, paints,inks, adhesives, and lubricants. One example of how the hydroxylation ofa glyceride may be performed is as follows: fat may be heated,preferably to about 30-50° C. combined with heptane and maintained attemperature for thirty minutes or more; acetic acid may then be added tothe mixture followed by an aqueous solution of sulfuric acid followed byan aqueous hydrogen peroxide solution which is added in small incrementsto the mixture over one hour; after the aqueous hydrogen peroxide, thetemperature may then be increased to at least about 60° C. and stirredfor at least six hours; after the stirring, the mixture is allowed tosettle and a lower aqueous layer formed by the reaction may be removedwhile the upper heptane layer formed by the reaction may be washed withhot water having a temperature of about 60° C.; the washed heptane layermay then be neutralized with an aqueous potassium hydroxide solution toa pH of about 5 to 7 and then removed by distillation under vacuum; thereaction product may then be dried under vacuum at 100° C. and the driedproduct steam-deodorized under vacuum conditions and filtered at about50° to 60° C. using diatomaceous earth.

Hydroxylation of microbial oils produced by the methods described hereincan be performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: U.S.Pat. Nos. 6,590,113 (Oil-based coatings and ink); 4,049,724(Hydroxylation process); 6,113,971 (Olive oil butter); 4,992,189(Lubricants and lube additives); 5,576,027 (Hydroxylated milk); and6,869,597 (Cosmetics).

Hydroxylated glycerolipids can be converted to estolides. Estolidesconsist of a glycerolipid in which a hydroxylated fatty acid constituenthas been esterified to another fatty acid molecule. Conversion ofhydroxylated glycerolipids to estolides can be carried out by warming amixture of glycerolipids and fatty acids and contacting the mixture witha mineral acid, as described by Isbell et al., JAOCS 71(2):169-174(1994). Estolides are useful in a variety of applications, includingwithout limitation those reported in the following: U.S. Pat. Nos.7,196,124 (Elastomeric materials and floor coverings); 5,458,795(Thickened oils for high-temperature applications); 5,451,332 (Fluidsfor industrial applications); 5,427,704 (Fuel additives); and 5,380,894(Lubricants, greases, plasticizers, and printing inks).

Another such chemical modification is olefin metathesis. In olefinmetathesis, a catalyst severs the alkylidene carbons in an alkene(olefin) and forms new alkenes by pairing each of them with differentalkylidine carbons. The olefin metathesis reaction provides a mechanismfor processes such as truncating unsaturated fatty acid alkyl chains atalkenes by ethenolysis, cross-linking fatty acids through alkenelinkages by self-metathesis, and incorporating new functional groups onfatty acids by cross-metathesis with derivatized alkenes.

In conjunction with other reactions, such as transesterification andhydrogenation, olefin metathesis can transform unsaturated glycerolipidsinto diverse end products. These products include glycerolipid oligomersfor waxes; short-chain glycerolipids for lubricants; homo- andhetero-bifunctional alkyl chains for chemicals and polymers; short-chainesters for biofuel; and short-chain hydrocarbons for jet fuel. Olefinmetathesis can be performed on triacylglycerols and fatty acidderivatives, for example, using the catalysts and methods reported inU.S. Pat. No. 7,119,216, US Patent Pub. No. 2010/0160506, and U.S.Patent Pub. No. 2010/0145086.

Olefin metathesis of bio-oils generally comprises adding a solution ofRu catalyst at a loading of about 10 to 250 ppm under inert conditionsto unsaturated fatty acid esters in the presence (cross-metathesis) orabsence (self-metathesis) of other alkenes. The reactions are typicallyallowed to proceed from hours to days and ultimately yield adistribution of alkene products. One example of how olefin metathesismay be performed on a fatty acid derivative is as follows: A solution ofthe first generation Grubbs Catalyst(dichloro[2(1-methylethoxy-α-O)phenyl]methylene-α-C](tricyclohexyl-phosphine) in toluene at a catalyst loading of 222 ppmmay be added to a vessel containing degassed and dried methyl oleate.Then the vessel may be pressurized with about 60 psig of ethylene gasand maintained at or below about 30° C. for 3 hours, wherebyapproximately a 50% yield of methyl 9-decenoate may be produced.

Olefin metathesis of oils produced by the methods described herein canbe performed in conjunction with one or more of the methods and/ormaterials, or to produce products, as reported in the following: PatentApp. PCT/US07/081,427 (α-olefin fatty acids) and U.S. patent applicationSer. Nos. 12/281,938 (petroleum creams), 12/281,931 (paintball guncapsules), 12/653,742 (plasticizers and lubricants), 12/422,096(bifunctional organic compounds), and 11/795,052 (candle wax).

Other chemical reactions that can be performed on microbial oils includereacting triacylglycerols with a cyclopropanating agent to enhancefluidity and/or oxidative stability, as reported in U.S. Pat. No.6,051,539; manufacturing of waxes from triacylglycerols, as reported inU.S. Pat. No. 6,770,104; and epoxidation of triacylglycerols, asreported in “The effect of fatty acid composition on the acrylationkinetics of epoxidized triacylglycerols”, Journal of the American OilChemists' Society, 79:1, 59-63, (2001) and Free Radical Biology andMedicine, 37:1, 104-114 (2004).

The generation of oil-bearing microbial biomass for fuel and chemicalproducts as described above results in the production of delipidatedbiomass meal. Delipidated meal is a byproduct of preparing algal oil andis useful as animal feed for farm animals, e.g., ruminants, poultry,swine and aquaculture. The resulting meal, although of reduced oilcontent, still contains high quality proteins, carbohydrates, fiber,ash, residual oil and other nutrients appropriate for an animal feed.Because the cells are predominantly lysed by the oil separation process,the delipidated meal is easily digestible by such animals. Delipidatedmeal can optionally be combined with other ingredients, such as grain,in an animal feed. Because delipidated meal has a powdery consistency,it can be pressed into pellets using an extruder or expander or anothertype of machine, which are commercially available.

The invention, having been described in detail above, is exemplified inthe following examples, which are offered to illustrate, but not tolimit, the claimed invention.

XIV. EXAMPLES Example 1 Fatty Acid Analysis by Fatty Acid Methyl EsterDetection

Lipid samples were prepared from dried biomass. 20-40 mg of driedbiomass was resuspended in 2 mL of 5% H₂SO₄ in MeOH, and 200 ul oftoluene containing an appropriate amount of a suitable internal standard(C19:0) was added. The mixture was sonicated briefly to disperse thebiomass, then heated at 70-75° C. for 3.5 hours. 2 mL of heptane wasadded to extract the fatty acid methyl esters, followed by addition of 2mL of 6% K₂CO₃ (aq) to neutralize the acid. The mixture was agitatedvigorously, and a portion of the upper layer was transferred to a vialcontaining Na₂SO₄ (anhydrous) for gas chromatography analysis usingstandard FAME GC/FID (fatty acid methyl ester gas chromatography flameionization detection) methods.

Example 2 Triacylglyceride Purification from Oil and Methods forTriacylglyceride Lipase Digestion

The triacylglyceride (TAG) fraction of each oil sample was isolated bydissolving ˜10 mg of oil in dichloromethane and loading it onto aBond-Elut aminopropyl solid-phase extraction cartridge (500 mg)preconditioned with heptane. TAGs were eluted with dicholoromethane-MeOH(1:1) into a collection tube, while polar lipids were retained on thecolumn. The solvent was removed with a stream of nitrogen gas. Trisbuffer and 2 mg porcine pancreatic lipase (Type II, Sigma, 100-400units/mg) were added to the TAG fraction, followed by addition of bilesalt and calcium chloride solutions. The porcine pancreatic lipasecleaves sn-1 and sn-3 fatty acids, thereby generating2-monoacylglycerides and free fatty acids. This mixture was heated withagitation at 40° C. for three minutes, cooled briefly, then quenchedwith 6 N HCl. The mixture was then extracted with diethyl ether and theether layer was washed with water then dried over sodium sulfate. Thesolvent was removed with a stream of nitrogen. To isolate themonoacylglyceride (MAG) fraction, the residue was dissolved in heptaneand loaded onto a second aminopropyl solid phase extraction cartridgepretreated with heptane. Residual TAGs were eluted with diethylether-dichloromethane-heptane (1:9:40), diacylglycerides (DAGs) wereeluted with ethyl acetate-heptane (1:4), and MAGs were eluted from thecartridge with dichloromethane-methanol (2:1). The resulting MAG, DAG,and TAG fractions were then concentrated to dryness with a stream ofnitrogen and subjected to routine direct transesterification method ofGC/FID analysis as described in Example 1.

Example 3 Engineering Microorganisms for Fatty Acid and Sn-2 ProfilesIncreased in Lauric Acid Through Exogenous LPAAT Expression

This example describes the use of recombinant polynucleotides thatencode a C. nucifera 1-acyl-sn-glycerol-3-phosphate acyltransferase (CnLPAAT) enzyme to engineer a microorganism in which the fatty acidprofile and the sn-2 profile of the transformed microorganism has beenenriched in lauric acid.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain A, was initially transformed with the plasmid construct pSZ1283according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1283, described inPCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696 herebyincorporated by reference, comprised the coding sequence of the Cupheawrightii FATB2 (CwTE2) thioesterase (SEQ ID NO: 10), 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe nuclear genome, and a S. cerevisiae suc2 sucrose invertase codingregion (SEQ ID NO: 4), to express the protein sequence given in SEQ IDNO: 3, under the control of C. reinhardtii β-tubulin promoter/5′UTR (SEQID NO: 5) and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO:6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO:7 and served as a selectable marker. The CwTE2 protein coding sequenceto express the protein sequence given in SEQ ID NO: 11, was under thecontrol of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C.vulgaris nitrate reductase 3′UTR. The protein coding regions of CwTE2and suc2 were codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

Upon transformation of pSZ1283 into Strain A, positive clones wereselected on agar plates with sucrose as the sole carbon source. Primarytransformants were then clonally purified and a single transformant,Strain B, was selected for further genetic modification. Thisgenetically engineered strain was transformed with plasmid constructpSZ2046 to interrupt the pLoop genomic locus of Strain B. ConstructpSZ2046 comprised the coding sequence of the C. nucifera1-acyl-sn-glycerol-3-phosphate acyltransferase (Cn LPAAT) enzyme (SEQ IDNO: 12), 5′ (SEQ ID NO: 13) and 3′ (SEQ ID NO: 14) homologousrecombination targeting sequences (flanking the construct) to the pLoopgenomic region for integration into the nuclear genome, and a neomycinresistance protein-coding sequence under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 5), and Chlorella vulgaris nitratereductase 3′ UTR (SEQ ID NO: 6). This NeoR expression cassette is listedas SEQ ID NO: 15 and served as a selectable marker. The Cn LPAAT proteincoding sequence was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′UTR.The protein coding regions of Cn LPAAT and NeoR were codon optimized toreflect the codon bias inherent in P. moriformis UTEX 1435 nuclear genesas described in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. The amino acid sequence of CnLPAAT is provided as SEQ ID NO: 16.

Upon transformation of pSZ2046 into Strain B, thereby generating StrainC, positive clones were selected on agar plates comprising G418.Individual transformants were clonally purified and grown at pH 7.0under conditions suitable for lipid production as detailed inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 (U1) grown on glucose as asole carbon source, untransformed Strain B and five pSZ2046 positivetransformants (Strain C, 1-5) are presented in Table 6.

TABLE 6 Effect of LPAAT expression on fatty acid profiles of transformedPrototheca moriformis (UTEX 1435) comprising a mid-chain preferringthioesterase. Area % Fatty acid U1 Strain B Strain C-1 Strain C-2 StrainC-3 Strain C-4 Strain C-5 C10:0 0.01 5.53 11.37 11.47 10.84 11.13 11.12C12:0 0.04 31.04 46.63 46.47 45.84 45.80 45.67 C14:0 1.27 15.99 15.1415.12 15.20 15.19 15.07 C16:0 27.20 12.49 7.05 7.03 7.30 7.20 7.19 C18:03.85 1.30 0.71 0.72 0.74 0.74 0.74 C18:1 58.70 24.39 10.26 10.41 10.9511.31 11.45 C18:2 7.18 7.79 7.05 6.93 7.30 6.88 7.01 C10-C12 0.50 36.5758.00 57.94 56.68 56.93 56.79

As shown in Table 6, the fatty acid profile of Strain B expressing CwTE2showed increased composition of C10:0, C12:0, and C14:0 fatty acids anda decrease in C16:0, C18:0, and C18:1 fatty acids relative to the fattyacid profile of the untransformed UTEX 1435 strain. The impact ofadditional genetic modification on the fatty acid profile of thetransformed strains, namely the expression of CnLPAAT in Strain B, is astill further increase in the composition of C10:0 and C12:0 fattyacids, a still further decrease in C16:0, C18:0, and C18:1 fatty acids,but no significant effect on the C14:0 fatty acid composition. Thesedata indicate that the CnLPAAT shows substrate preference in the contextof a microbial host organism.

The untransformed P. moriformis (UTEX 1435) is characterized by a fattyacid profile comprising less than 0.5% C12 fatty acids and less than 1%C10-C12 fatty acids. In contrast, the fatty acid profile of Strain Bexpressing a C. wrightii thioesterase comprised 31% C12:0 fatty acids,with C10-C12 fatty acids comprising greater than 36% of the total fattyacids. Further, fatty acid profiles of Strain C, expressing a higherplant thioesterase and a CnLPAAT enzyme, comprised between 45.67% and46.63% C12:0 fatty acids, with C10-C12% fatty acids comprising between71 and 73% of total fatty acids. The result of expressing an exogenousthioesterase was a 62-fold increase in the percentage of C12 fatty acidpresent in the engineered microbe. The result of expressing an exogenousthioesterase and exogenous LPAAT was a 92-fold increase in thepercentage of C12 fatty acids present in the engineered microbe.

The TAG fraction of oil samples extracted from Strains A, B, and C wereanalyzed for the sn-2 profile of their triacylglycerides. The TAGs wereextracted and processed as described in Example 2 and analysed as inExamples 1 and 2. The fatty acid composition and the sn-2 profiles ofthe TAG fraction of oil extracted from Strains A, B, and C (expressed asArea % of total fatty acids) are presented in Table 7. Values notreported are indicated as “n.r.”

TABLE 7 Effect of LPAAT expression on the fatty acid composition and thesn-2 profile of TAGs produced from transformed Prototheca moriformis(UTEX 1435) comprising a mid-chain preferring thioesterase. Strain C(pSZ1500 + Strain Strain A (untransformed) Strain B (pSZ1500) pSZ2046)Area % fatty sn-2 sn-2 sn-2 acid FA profile FA profile FA profile C10:0n.r. n.r. 11.9 14.2 12.4 7.1 C12:0 n.r. n.r. 42.4 25 47.9 52.8 C14:0 1.00.6 12 10.4 13.9 9.1 C16:0 23.9 1.6 7.2 1.3 6.1 0.9 C18:0 3.7 0.3 n.rn.r. 0.8 0.3 C18:1 64.3 90.5 18.3 36.6 9.9 17.5 C18:2 4.5 5.8 5.8 10.86.5 10 C18:3 n.r. n.r. n.r. n.r. 1.1 1.6

As shown in Table 7, the fatty acid composition of triglycerides (TAGs)isolated from Strain B expressing CwTE2 was increased for C10:0, C12:0,and C14:0 fatty acids and decrease in C16:0 and C18:1 fatty acidsrelative to the fatty acid profile of TAGs isolated from untransformedStrain A. The impact of additional genetic modification on the fattyacid profile of the transformed strains, namely the expression ofCnLPAAT, was a still further increase in the composition of C10:0 andC12:0 fatty acids, a still further decrease in C16:0, C18:0, and C18:1fatty acids, but no significant effect on the C14:0 fatty acidcomposition. These data indicate that expression of the exogenousCnLPAAT improves the midchain fatty acid profile of transformedmicrobes.

The untransformed P. moriformis (UTEX 1435) Strain A is characterized byan sn-2 profile of about 0.6% C14, about 1.6% C16:0, about 0.3% C18:0,about 90% C18:1, and about 5.8% C18:2. In contrast to Strain A, StrainB, expressing a C. wrightii thioesterase is characterized by an sn-2profile that is higher in midchain fatty acids and lower in long chainfatty acids. C12 fatty acids comprised 25% of the sn-2 profile of StrainB. The impact of additional genetic modification on the sn-2 profile ofthe transformed strains, namely the expression of CnLPAAT, was still afurther increase in C12 fatty acids (from 25% to 52.8%), a decrease inC18:1 fatty acids (from 36.6% to 17.5%), and a decrease in C10:0 fattyacids. (The sn-2 profile composition of C14:0 and C16:0 fatty acids wasrelatively similar for Strains B and C.)

These data demonstrate the utility and effectiveness of polynucleotidespermitting exogenous LPAAT expression to alter the fatty acid profile ofengineered microorganisms, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids in microbial cells. Thesedata further demonstrate the utility and effectiveness ofpolynucleotides permitting exogenous thioesterase and exogenous LPAATexpression to alter the sn-2 profile of TAGs produced by microbialcells, in particular in increasing the C12 composition of sn-2 profilesand decreasing the C18:1 composition of sn-2 profiles.

Example 4 Thermal Behavior of Oils Produced from Recombinant Microalgae

FIGS. 1-14 include fatty acid profiles and melting curves of refined,bleached and deodorized oils from genetically engineered Protothecamoriformis strains. In some cases, modifications of the melting curvesare obtained via genetic engineering. For example, some of the oilsproduced have shallower or sharper melting transitions relative tocontrol microalgal oils (i.e., those produced from strains lacking agiven genetic modification) or relative to widely available plant oils.In addition, FIG. 12 shows scanning calorimetry for a high palmitic oilwhen tempered by holding at room temperature for several days (lowertrace) and for the same oil after performing the first scan (uppertrace). The scans ranged from −60° C. to +50° C. with a heating rate of10° C./minute. The differences between the two traces suggests thattempering of the oil caused a change in crystal structure within theoil.

Also of note, FIGS. 10 and 11 show stability testing of RBD-5 and RBD 6.Remarkably, RBD-6, an oil with less than 0.1% 18:2 and 18:3 fatty acidswas substantially stable as measured by the oxidative stability index(AOCS Method Cd 12b-92) even after 36 hours of heating at 110° C.

Table 8, below, gives details of the genetic engineering of the strainsidentified in FIGS. 1-13.

TABLE 8 Genetically engineered strains. RB Z Ulmus Americanathioesterase RBD-1 Cuphea wrightii FATB2 thioesterase driven by amt03RBD-2 Ulmus americana thioesterase RBD-3 Native C. hookerianaC16:0-specific thioesterase with amt03 promoter RBD Y Ulmus Americanathioesterase with Btub promoter RBD X SAD2B knockout with native Cwrightii FAT2B thioesterase, amt03 promoter RBD W SAD2B KO with NativeC. wrightii FATB2 driven by amt03 at insertion site RBD-4 control strainRBD-5 FATA-1 knockout with Carthamus oleate sp. TE driven by amt03promoter at insertion site RBD-6 FADc knockout with Carthamus tinctoriusoleoyl thioesterase

Example 5 Characteristics of Processed Oil Produced from EngineeredMicroorganisms

Methods and effects of transforming Prototheca moriformis (UTEX 1435)with transformation vector pSZ1500 (SEQ ID NO: 17) have been previouslydescribed in PCT Application Nos. PCT/US2011/038463, PCT/US2011/038464,and PCT/US2012/023696.

A classically mutagenized (for higher oil production) derivative ofProtetheca moriformis (UTEX 1435), Strain A, was transformed withpSZ1500 according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1500 comprised nucleotidesequence of the Carthamus tinctorius oleyl-thioesterase (CtOTE) gene,codon-optimized for expression in P. moriformis UTEX 1435. The pSZ1500expression construct included 5′ (SEQ ID NO: 18) and 3′ (SEQ ID NO: 19)homologous recombination targeting sequences (flanking the construct) tothe FADc genomic region for integration into the nuclear genome and a S.cerevisiae suc2 sucrose invertase coding region under the control of C.reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselection marker. The CtOTE coding region was under the control of theP. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgarisnitrate reductase 3′UTR, and the native transit peptide was replacedwith the C. protothecoides stearoyl-ACP desaturase transit peptide (SEQID NO: 9). The protein coding regions of CtOTE and suc2 were codonoptimized to reflect the codon bias inherent in P. moriformis UTEX 1435nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ1500 transformants of Strain A were selected on agar platescontaining sucrose as a sole carbon source, clonally purified, and asingle engineered line, Strain D was selected for analysis. Strain D wasgrown as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Hexaneextraction of the oil from the generated biomass was then performedusing standard methods, and the resulting triglyceride oil wasdetermined to be free of residual hexane. Other methods of extraction ofoil from microalgae using an expeller press are described in PCTApplication No. PCT/US2010/031108 and are hereby incorporated byreference.

Different lots of oil extracted from biomass of Strain D were refined,bleached, and deodorized using standard vegetable oil processingmethods. These procedures generated oil samples RBD437, RBD469, RBD501,RBD 502, RBD503, and RBD529, which were subjected to analytical testingprotocols according to methods defined through the American OilChemists' Society, the American Society for Testing and Materials, andthe International Organization for Standardization. The results of theseanalyses are summarized below in Tables 9-14.

TABLE 9 Analytical results for oil sample RBD469. Method Number TestDescription Results Units AOCS Ca 3a-46 Insoluble impurities <0.01 %AOCS Ca 5a-40 Free Fatty Acids (Oleic) 0.02 % AOCS Ca 5a-40 Acid Value0.04 mg KOH/g AOCS CA 9f-57 Neutral oil 98.9 % D97 Cloud Point −15 degC. D97 Pour Point −18 deg C. Karl Fischer Moisture 0.01 % AOCS Cc 13d-55Chlorophyll <0.01 ppm (modified) Iodine Value 78.3 g I₂/100 g AOCS Cd8b-90 Peroxide Value 0.31 meq/kg ISO 6885 p-Anisidine Value 0.65 AOCS Cc18-80 Dropping Melting point 6.2 deg C. (Mettler) AOCS Cd 11d-96Tricylglicerides 98.6 % AOCS Cd 11d-96 Monoglyceride <0.01 % AOCS Cd11d-96 Diglicerides 0.68 % AOCS Cd 20-91 Total Polar Compounds 2.62 %IUPAC, 2.507 and Oxidized & Polymerized 17.62 % 2.508 TricylgliceridesAOCS Cc 9b-55 Flash Point 244 deg C. AOCS Cc 9a-48 Smoke Point 232 degC. AOCS Cd 12b-92 Oxidataive Stability Index 31.6 hours Rancimat (110°C.) AOCS Ca 6a-40 Unsaponified Matter 2.28 %

RBD469 oil was analyzed for trace element content, solid fat content,and Lovibond color according to AOCS methods. Results of these analysesare presented below in Table 10, Table 10, and Table 11.

TABLE 10 ICP Elemental Analysis of RBD469 oil. Method Number TestDescription Results in ppm AOCS Ca 20-99 and Phosphorus 1.09 AOCS Ca17-01 Calcium 0.1 (modified) Magnesium 0.04 Iron <0.02 Sulfur 28.8Copper <0.05 Potassium <0.50 Sodium <0.50 Silicon 0.51 Boron 0.06Aluminum <0.20 Lead <0.20 Lithium <0.02 Nickel <0.20 Vanadium <0.05 Zinc<0.02 Arsenic <0.20 Mercury <0.20 Cadmium <0.03 Chromium <0.02 Manganese<0.05 Silver <0.05 Titanium <0.05 Selenium <0.50 UOP779 Chloride organic<1 UOP779 Chloride inorganic 7.24 AOCS Ba 4e-93 Nitrogen 6.7

TABLE 11 Solid Fat Content of RBD469 Oil Method Number Solid Fat ContentResult AOCS Cd 12b-93 Solid Fat Content 10° C. 0.13% AOCS Cd 12b-93Solid Fat Content 15° C. 0.13% AOCS Cd 12b-93 Solid Fat Content 20° C.0.28% AOCS Cd 12b-93 Solid Fat Content 25° C. 0.14% AOCS Cd 12b-93 SolidFat Content 30° C. 0.08% AOCS Cd 12b-93 Solid Fat Content 35° C. 0.25%

TABLE 12 Lovibond Color of RBD469 Oil Method Number Color Result UnitAOCS Cc 13j-97 red 2 Unit AOCS Cc 13j-97 yellow 27 Unit

RBD469 oil was subjected to transesterification to produce fatty acidmethyl esters (FAMEs). The resulting FAME profile of RBD469 is shown inTable 12.

TABLE 13 FAME Profile of RBD469 Oil Fatty Acid Area % C10 0.01 C12:00.04 C14:0 0.64 C15:0 0.08 C16:0 8.17 C16:1 iso 0.39 C16:1 0.77 C17:00.08 C18:0 1.93 C18:1 85.88 C18:1 iso 0.05 C18:2 0.05 C20:0 0.3 C20:10.06 C20:1 0.44 C22:0 0.11 C23:0 0.03 C24:0 0.1 Total FAMEs Identified99.13

The oil stability indexes (OSI) of 6 RBD oil samples withoutsupplemented antioxidants and 3 RBD oil samples supplemented withantioxidants were analyzed according to the Oil Stability Index AOCSMethod Cd 12b-92. Shown in Table 14 are the results of OSI AOCS Cd12b-92 tests, conducted at 110° C., performed using a Metrohm 873Biodiesel Rancimat. Results, except where indicated with an astericks(*), are the average of multiple OSI runs. Those samples not analyzedare indicated (NA).

TABLE 14 Oil Stability Index at 110° C. of RBD oil samples with andwithout antioxidants. Antioxidant Antioxidant OSI (hours) for each RBDSample added Concentration RBD437 RBD469 RBD502 RBD501 RBD503 RBD529None 0 65.41 38.33 72.10 50.32 63.04 26.68 Tocopherol 35 ppm/ 77.7248.60 82.67 NA NA NA & Ascorbyl 16.7 ppm Palmitate Tocopherol 140 ppm/130.27 81.54* 211.49* NA NA NA & Ascorbyl 66.7 ppm Palmitate Tocopherol1050 ppm/ >157*  >144 242.5* NA NA NA & Ascorbyl 500 ppm PalmitateTocopherol 50 ppm NA 46.97 NA NA NA NA TBHQ 20 ppm 63.37 37.4 NA NA NANA

The untransformed P. moriformis (UTEX 1435) acid profile comprises lessthan 60% C18:1 fatty acids and greater than 7% C18:2 fatty acids. Incontrast, Strain D (comprising pSZ1500) exhibited fatty acid profileswith an increased composition of C18:1 fatty acids (to above 85%) and adecrease in C18:2 fatty acids (to less than 0.06%). Upon refining,bleaching, and degumming, RBD oils samples prepared from the oil madefrom strain E exhibited OSI values>26 hrs. With addition ofantioxidants, the OSI of RBD oils prepared from oils of Strain Dincreased from 48.60 hours to greater than 242 hours. In otherexperiments, OSI values of over 400 hours were achieved. Additionalproperties of a low polyunsaturated oil according to embodiments of theinvention are given in FIG. 16.

Example 6 Improving the Levels of Oleic Acid of Engineered MicrobesThrough Allelic Disruption of a Fatty Acid Desaturase and an Acyl-ACPThioesterase

This example describes the use of a transformation vector to disrupt aFATA locus of a Prototheca moriformis strain previously engineered forhigh oleic acid and low linoleic acid production. The transformationcassette used in this example comprised a selectable marker andnucleotide sequences encoding a P. moriformis KASII enzyme to engineermicroorganisms in which the fatty acid profile of the transformedmicroorganism has been altered for further increased oleic acid andlowered palmitic acid levels.

Strain D, described in Example 5 and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 17) according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strainwas used as the host for transformation with construct pSZ2276 toincrease expression of a KASII enzyme while concomitantly ablating anendogenous acyl-ACP thioesterase genetic locus to generate Strain E. ThepSZ2276 transformation construct included 5′ (SEQ ID NO: 20) and 3′ (SEQID NO: 21) homologous recombination targeting sequences (flanking theconstruct) to the FATA1 genomic region for integration into the P.moriformis nuclear genome, an A. thaliana THIC protein coding regionunder the control of the C. protothecoides actin promoter/5′UTR (SEQ IDNO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). ThisAtTHIC expression cassette is listed as SEQ ID NO: 23 and served as aselection marker. The P. moriformis KASII protein coding region wasunder the control of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO:8) and C. vulgaris nitrate reductase 3′UTR, and the native transitpeptide of the KASII enzyme was replaced with the C. protothecoidesstearoyl-ACP desaturase transit peptide (SEQ ID NO: 9). Thecodon-optimized sequence of PmKASII comprising a C. protothecoides S106stearoyl-ACP desaturase transit peptide is provided the sequencelistings as SEQ ID NO: 24. SEQ ID NO: 25 provides the proteintranslation of SEQ ID NO: 24. The protein coding regions of PmKASII andsuc2 were codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

Primary pSZ2276 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and a single engineered line,strain E was selected for analysis. Strain E was cultivated underheterotrophic lipid production conditions at pH5.0 and pH7.0 asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) from the transgenic line arising from transformationwith pSZ2276 into Strain D are shown in Table 15.

TABLE 15 Fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and E engineered for increased oleic acid and loweredlinoleic acid levels. Transformation Area % Fatty Acid StrainConstruct(s) pH C16:0 C18:0 C18:1 C18:2 C20:1 Strain A None pH 5 26.63.3 60.5 6.7 0.07 Strain A None pH 7 28.3 4.1 58 6.5 0.06 Strain DpSZ1500 pH 5 17 3.6 77.1 0.01 0.14 Strain D pSZ1500 pH 7 19.5 5.3 72.60.01 0.09 Strain E pSZ1500 + pH 5 4.1 2.36 88.5 0.04 3.1 pSZ2276 StrainE pSZ1500 + pH 7 2.1 7.8 87.9 0.01 0.5 pSZ2276

As shown in Table 15, targeted interruption of FADc alleles with a CtOTEexpression cassette impacted the fatty acid profiles of transformedmicroorganisms. Fatty acid profiles of Strain D (comprising the pSZ1500transformation vector) showed increased composition of C18:1 fatty acidswith a concomitant decrease in C16:0 and C18:2 fatty acids relative toStrain A. Subsequent transformation of Strain D with pSZ2276 tooverexpress a P. moriformis (UTEX 1435) KASII protein whileconcomitantly ablating a FATA genetic locus (thereby generating StrainE) resulted in still further impact on the fatty acid profiles of thetransformed microorganisms. Fatty acid profiles of Strain E showedincreased composition of C18:1 fatty acids, with a further decrease inC16:0 fatty acids relative to Strains A and D. Propagation of Strain Ein culture conditions at pH 7, to induce expression from the Amt03promoter, resulted in a fatty acid profile that was higher in C18:0 andC18:1 fatty acids and lower in C16:0 fatty acids, relative to the samestrain cultured at pH 5.

These data demonstrate the utility of multiple genetic modifications toimpact the fatty acid profile of a host organism for increased levels ofoleic acid with concomitant decreased levels of linoleic acid andpalmitic acid. Further, this example illustrates the use of recombinantpolynucleotides to target gene interruption of an endogenous FATA allelewith a cassette comprising a pH-regulatable promoter to controlexpression of an exogenous KASII protein-coding region in order to alterthe fatty acid profile of a host microbe.

Example 7 Conditional Expression of a Fatty Acid Desaturase

This example describes the use of a transformation vector toconditionally express a delta 12 fatty acid desaturase (FADs) in aPrototheca moriformis strain previously engineered for high oleic acidand very low linoleic acid production in both seed and lipidproductivity stages of propagation. Very low linoleic acid levels innatural oils are sought for use in certain applications. However,absence of linoleic acid during cell division phase (“seed stage”) of ahost microbe is disadvantageous. Linoleic acid may be supplemented tothe seed medium to hasten cell division and not added during lipidproduction, but this addition imposes unwanted costs. To overcome thischallenge, a transformation cassette was constructed for regulatedexpression of a FAD2 enzyme such that levels of linoleic acidssufficient for cell division could be achieved and oil with very lowlevels of linoleic acids could be produced during the oil productionphase of culture of a microorgansim. The transformation cassette used inthis example comprised a selectable marker, a pH-regulatable promoter,and nucleotide sequences encoding a P. moriformis FAD2 enzyme toengineer microorganisms in which the fatty acid profile of thetransformed microorganism has been altered for increased oleic acidproduction and regulatable linoleic acid production.

Strain D, described in Examples 5, 6, and in PCT/US2012/023696, is aclassically mutagenized (for higher oil production) derivative of P.moriformis (UTEX 1435) subsequently transformed with the transformationconstruct pSZ1500 (SEQ ID NO: 17) according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. This strainwas used as the host for transformation with construct pSZ2413 tointroduce a pH-driven promoter for regulation of a P. moriformis FAD2enzyme. The pSZ2413 transformation construct included 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe P. moriformis nuclear genome, an A. thaliana THIC protein codingregion under the control of the C. protothecoides actin promoter/5′UTR(SEQ ID NO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6).This AtTHIC expression cassette is listed as SEQ ID NO: 23 and served asa selection marker. The P. moriformis FAD2 protein coding region wasunder the control of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO:8) and C. vulgaris nitrate reductase 3′UTR. The codon-optimized sequenceof PmFAD2 is provided the sequence listings as SEQ ID NO: 26. SEQ ID NO:27 provides the protein translation of SEQ ID NO: 26. The protein codingregions of PmFAD2 and suc2 were codon optimized to reflect the codonbias inherent in P. moriformis UTEX 1435 nuclear genes as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ2413 transformants of Strain D were selected on agar plateslacking thiamine, clonally purified, and isolates of the engineeredline, Strain F were selected for analysis. These isolates werecultivated under heterotrophic lipid production conditions at pH7.0 (toactivate expression of FAD2 from the PmAmt03 promoter) and at pH5.0, asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The resulting profile of C18:2 fatty acids(expressed in Area %) from nine representative isolates of transgenicStrain F (F-1 through F-9) arising from transformation with pSZ2413 intoStrain D are shown in Table 16.

TABLE 16 C18:2 fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains A, D, and F. Area % C18:2 Transformation pH pH Strain Construct(s) 5.0 7.0 A None 6.07 7.26 D pSZ1500 0.01 0.01 F-1 pSZ1500 + pSZ24130.37 5.29 F-2 pSZ1500 + pSZ2413 0.45 6.87 F-3 pSZ1500 + pSZ2413 0.506.79 F-4 pSZ1500 + pSZ2413 0.57 5.06 F-5 pSZ1500 + pSZ2413 0.57 7.58 F-6pSZ1500 + pSZ2413 0.60 6.88 F-7 pSZ1500 + pSZ2413 0.62 6.52 F-8pSZ1500 + pSZ2413 0.63 5.79 F-9 pSZ1500 + pSZ2413 0.77 4.53

As shown in Table 16 the impact of regulated expression of the PmFAD2enzyme, effected though strain culture at different pH levels, is aclear increase in the composition of C18:2 fatty acids in thetransformed microorganism. Linoleic acid comprises about 6% to about7.3% of fatty acids of Strain A. In contrast, Strain D (comprising thepSZ1500 transformation vector to ablate both FAD2 alleles) ischaracterized by a fatty acid profile of 0.01% linoleic acid.Transformation of Strain D with pSZ2413 to generate Strain F results ina recombinant microbe in which the production of linoleic acid isregulated by the Amt03 promoter. Propagation of Strain F isolates inculture conditions at pH 7, to induce FAD2 expression from the Amt03promoter, resulted in a fatty acid profile characterized by about 4.5%to about 7.5% linoleic acid. In contrast, propagation of Strain Fisolates in culture conditions at pH 5 resulted in a fatty acid profilecharacterized by about 0.33 to about 0.77% linoleic acid.

These data demonstrate the utility of and effectiveness of recombinantpolynucleotides permitting conditional expression of a FAD2 enzyme toalter the fatty acid profile of engineered microorganisms, and inparticular in regulating the production of C18:2 fatty acids inmicrobial cells.

Example 8 Analysis of Regiospecific Profile

LC/MS TAG distribution analyses were carried out using a Shimadzu Nexeraultra high performance liquid chromatography system that included aSIL-30AC autosampler, two LC-30AD pumps, a DGU-20A5 in-line degasser,and a CTO-20A column oven, coupled to a Shimadzu LCMS 8030 triplequadrupole mass spectrometer equipped with an APCI source. Data wasacquired using a Q3 scan of m/z 350-1050 at a scan speed of 1428 u/secin positive ion mode with the CID gas (argon) pressure set to 230 kPa.The APCI, desolvation line, and heat block temperatures were set to 300,250, and 200° C., respectively, the flow rates of the nebulizing anddrying gases were 3.0 L/min and 5.0 L/min, respectively, and theinterface voltage was 4500 V. Oil samples were dissolved indichloromethane-methanol (1:1) to a concentration of 5 mg/mL, and 0.8 μLof sample was injected onto Shimadzu Shim-pack XR-ODS III (2.2 μm,2.0×200 mm) maintained at 30° C. A linear gradient from 30%dichloromethane-2-propanol (1:1)/acetonitrile to 51%dichloromethane-2-propanol (1:1)/acetonitrile over 27 minutes at 0.48mL/min was used for chromatographic separations.

Example 9 Engineering Microbes for Increased Production of SOS, POP, andPOS Triacylglycerides

This example describes the use of recombinant polynucleotides thatencode a C18:0-preferring Brassica napus thioesterase (BnOTE) enzyme toengineer a microorganism in which the triacylglyceride distribution ofthe transformed microorganism has been enriched in SOS, POS, and POPtriacylglycerides.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain A, was initially transformed with the plasmid construct pSZ1358according to biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. pSZ1358, described inPCT/US2012/023696, hereby incorporated by reference, comprised thecoding sequence of the Brassica napus thioesterase (BnOTE) thioesterase(SEQ ID NO: 28), 5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2) homologousrecombination targeting sequences (flanking the construct) to the 6Sgenomic region for integration into the nuclear genome, and a S.cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), toexpress the protein sequence given in SEQ ID NO: 3, under the control ofC. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselectable marker. The BnOTE protein coding sequence to express theprotein sequence given in SEQ ID NO: 29, was under the control of the P.moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitratereductase 3′UTR. The protein coding regions of BnOTE and suc2 were codonoptimized to reflect the codon bias inherent in P. moriformis UTEX 1435nuclear genes as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696.

Primary pSZ1358 transformants of Strain A were selected on agar platescontaining sucrose as a sole carbon source, clonally purified, andsingle engineered line, Strain G was selected for analysis. Strain G wascultivated under heterotrophic lipid production conditions at pH7.0 (toactivate expression of BnOTE from the PmAmt03 promoter) as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Oil samples obtained fromStrain A and Strain G were analyzed for fatty acid composition usingmethods described in Examples 1 and 2, and, using the methods describedin Example 8, for the regiospecificity of triacylglcyerides in the oil.Fatty acid profiles of TAGs isolated from Strain A and G are shown inTable 17. Table 18 presents the regiospecificity profile of POP, POS,and SOS TAGs present in oil samples from Strain A and G.

TABLE 17 Effect of BnOTE expression on the fatty acid composition andthe sn-2 profile of TAGs produced from transformed Protothecamoriformis. Strain G Area % Strain A (pSZ1358) Fatty FA FA acid profileprofile C10:0 n.r. 0.5 C12:0 n.r. 0.5 C14:0 1.0 1.3 C16:0 23.9 25.8C18:0 3.7 30.4 C18:1 64.3 30.2 C18:2 4.5 8.8 C18:3 α n.r. 0.4

TABLE 18 Effect of BnOTE expression on the regiospecific profile of POP,POS, and SOS TAGs produced from transformed Prototheca moriformis.Strain A (untransformed) Strain G (pSZ1358) normalized normalized TAGArea % area % Area % area % POP 13.09 76.8 10.6 23.5 POS 3.51 20.5 21.046.6 SOS 0.45 2.6 13.5 29.9 total 17.05 100 45.0 100

As shown in Table 17, the fatty acid composition of TAGs isolated fromStrain G expressing BnOTE was markedly increased for C18:0 fatty acids(from 3.7% to 30.4%) and decreased in C18:1 fatty acids (from 64.3% to30.2%) relative to the fatty acid profile of TAGs isolated fromuntransformed Strain A. The fatty acid composition of TAGs isolated fromStrain A was characterized by about 23.9% palmitic acid, 3.7% stearicacid, and 64.3% oleic acid, a ratio for P:S:O of about 6.5:1:17.4. Incontrast, the fatty acid composition of TAGs isolated from Strain G wascharacterized by about 25.8% palmitic acid, 30.4% stearic acid, and30.2% oleic acid, a ratio for P:O:S of about 1:1.18:1.17.

The impact of expression of a C18:0 preferring thioesterease on theregiospecific profile of POP, POS, and SOS TAGs of oils produced fromthe transformed microorganism was an increase in all three TAGs as aproportion of the total TAGs present in the oil. As shown in Table 18,the sum of POP+POS+SOS TAGs accounted for 45% of the TAGs produced byStrain G, whereas POP, POS, and SOS TAGs summed to only about 17% ofTAGs produced in Strain A.

These data demonstrate the utility and effectiveness of polynucleotidespermitting exogenous thioesterase expression to alter the fatty acid andregiospecific profiles of TAGs of engineered microorganisms, inparticular to increase the distribution of POP, POS, and SOS TAGs.

Examples 10-33 Engineering of Microorganisms

Examples 10-33 below describe the engineering of various microorganismsin accordance with the present invention. To alter the fatty acidprofile of a microorganism, microorganisms can be genetically modifiedwherein endogenous or exogenous lipid biosynthesis pathway enzymes areexpressed, overexpressed, or attenuated. Steps to genetically engineer amicrobe to alter its fatty acid profile as to the degree of fatty acidunsaturation and to decrease or increase fatty acid chain lengthcomprise the design and construction of a transformation vector (e.g., aplasmid), transformation of the microbe with one or more vectors,selection of transformed microbes (transformants), growth of thetransformed microbe, and analysis of the fatty acid profile of thelipids produced by the engineered microbe.

Transgenes that alter the fatty acid profiles of host organisms can beexpressed in numerous eukaryotic microbes. Examples of expression oftransgenes in eukaryotic microbes including Chlamydomonas reinhardtii,Chlorella ellipsoidea, Chlorella saccarophila, Chlorella vulgaris,Chlorella kessleri, Chlorella sorokiniana, Haematococcus pluvialis,Gonium pectorale, Volvox carteri, Dunaliella tertiolecta, Dunaliellaviridis, Dunaliella salina, Closterium peracerosum-strigosum-littoralecomplex, Nannochloropsis sp., Thalassiosira pseudonana, Phaeodactylumtricornutum, Navicula saprophila, Cylindrotheca fusiformis, Cyclotellacryptica, Symbiodinium microadriacticum, Amphidinium sp., Chaetocerossp., Mortierella alpina, and Yarrowia lipolytica can be found in thescientific literature. These expression techniques can be combined withthe teachings of the present invention to produce engineeredmicroorganisms with altered fatty acid profiles.

Transgenes that alter the fatty acid profiles of host organisms or alterthe regiospecific distribution of glycerolipds produced by hostorganisms can also be expressed in numerous prokaryotic microbes.Examples of expression of transgenes in oleaginous microbes includingRhodococcus opacus can be found in the literature. These expressiontechniques can be combined with the teachings of the present inventionto produce engineered microorganisms with altered fatty acid profiles.

TABLE 19A-D Codon preference listing. Closterium peracerosum- strigosum-Amino Chlorella Chlorella Chlorella Chlorella Dunaliella VolvoxHaematococcus littorale Acid Codon sorokiniana vulgaris ellipsoideakessleri tertiolecta carteri pluvialis complex Ala GCG 0.20 0.25 0.150.14 0.09 0.25 0.21 0.48 Ala GCA 0.05 0.24 0.32 0.10 0.17 0.13 0.27 0.10Ala GCT 0.12 0.16 0.26 0.18 0.31 0.26 0.17 0.15 Ala GCC 0.63 0.35 0.270.58 0.43 0.36 0.35 0.26 Arg AGG 0.03 0.09 0.10 0.09 0.26 0.08 0.14 0.04Arg AGA 0.04 0.05 0.14 0.01 0.09 0.03 0.05 0.00 Arg CGG 0.06 0.19 0.090.06 0.06 0.17 0.15 0.18 Arg CGA 0.00 0.10 0.08 0.00 0.08 0.08 0.10 0.00Arg CGT 0.06 0.09 0.37 0.14 0.12 0.22 0.13 0.13 Arg CGC 0.81 0.48 0.220.71 0.40 0.43 0.42 0.64 Asn AAT 0.04 0.16 0.43 0.06 0.27 0.23 0.21 0.04Asn AAC 0.96 0.84 0.57 0.94 0.73 0.77 0.79 0.96 Asp GAT 0.13 0.25 0.470.12 0.40 0.35 0.27 0.30 Asp GAC 0.87 0.75 0.53 0.88 0.60 0.65 0.73 0.70Cys TGT 0.06 0.13 0.43 0.09 0.20 0.17 0.27 0.06 Cys TGC 0.94 0.87 0.570.91 0.80 0.83 0.64 0.94 End TGA 0.00 0.72 0.14 0.14 0.36 0.24 0.70 0.75End TAG 0.33 0.11 0.29 0.00 0.00 0.18 0.22 0.00 End TAA 0.67 0.17 4.000.86 0.64 0.59 0.09 0.25 Gln CAG 0.42 0.40 0.15 0.40 0.27 0.29 0.33 0.53Gln CAA 0.04 0.04 0.21 0.40 0.27 0.07 0.10 0.09 Glu GAG 0.53 0.50 0.330.40 0.27 0.53 0.49 0.31 Glu GAA 0.02 0.06 0.31 0.40 0.27 0.11 0.07 0.06Gly GGG 0.04 0.16 0.19 0.08 0.10 0.12 0.22 0.31 Gly GGA 0.02 0.11 0.130.07 0.13 0.12 0.11 0.06 Gly GGT 0.03 0.12 0.39 0.24 0.25 0.23 0.15 0.09Gly GGC 0.91 0.61 0.29 0.96 0.51 0.53 0.52 0.53 His CAT 0.14 0.16 0.300.08 0.25 0.35 0.27 0.33 His CAC 0.86 0.84 0.70 0.93 0.75 0.65 0.73 0.67Ile ATA 0.00 0.04 0.07 0.01 0.04 0.08 0.09 0.03 Ile ATT 0.15 0.30 0.630.29 0.31 0.35 0.29 0.23 Ile ATC 0.85 0.66 0.65 0.69 0.65 0.57 0.62 0.74Leu TTG 0.03 0.07 0.03 0.05 0.14 0.14 0.16 0.04 Leu TTA 0.00 0.01 0.320.00 0.02 0.03 0.02 0.00 Leu CTG 0.72 0.61 0.34 0.61 0.60 0.45 0.53 0.31Leu CTA 0.01 0.03 0.03 0.04 0.04 0.07 0.07 0.01 Leu CTT 0.04 0.08 0.160.06 0.06 0.14 0.09 0.04 Leu CTC 0.20 0.20 0.12 0.24 0.14 0.17 0.13 0.60Lys AAG 0.98 0.94 0.54 0.98 0.90 0.90 0.84 0.86 Lys AAA 0.02 0.06 0.460.02 0.10 0.10 0.16 0.14 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Phe TTT 0.28 0.32 0.42 0.31 0.24 0.27 0.35 0.09 Phe TTC 0.72 0.68 0.580.69 0.76 0.73 0.65 0.91 Pro CCG 0.18 0.31 0.09 0.07 0.04 0.34 0.15 0.28Pro CCA 0.06 0.17 0.36 0.07 0.04 0.20 0.24 0.15 Pro CCT 0.10 0.14 0.250.17 0.04 0.19 0.29 0.12 Pro CCC 0.66 0.38 0.29 0.69 0.04 0.27 0.32 0.44Ser AGT 0.03 0.04 0.14 0.02 0.08 0.08 0.07 0.04 Ser AGC 0.27 0.38 0.180.18 0.31 0.27 0.31 0.05 Ser TCG 0.12 0.14 0.08 0.10 0.02 0.19 0.10 0.22Ser TCA 0.03 0.08 0.14 0.08 0.09 0.09 0.14 0.16 Ser TCT 0.09 0.11 0.260.18 0.19 0.14 0.13 0.05 Ser TCC 0.47 0.24 0.20 0.44 0.30 0.24 0.24 0.47Thr ACG 0.11 0.20 0.13 0.05 0.12 0.27 0.19 0.30 Thr ACA 0.01 0.20 0.320.07 0.20 0.12 0.23 0.06 Thr ACT 0.12 0.13 0.29 0.12 0.24 0.20 0.18 0.22Thr ACC 0.76 0.47 0.26 0.76 0.44 0.41 0.40 0.42 Trp TGG 1.00 1.00 1.001.00 1.00 1.00 1.00 1.00 Tyr TAT 0.07 0.15 0.43 0.27 0.28 0.24 0.19 0.07Tyr TAC 0.93 0.85 0.57 0.73 0.72 0.76 0.81 0.93 Val GTG 0.71 0.54 0.370.60 0.54 0.46 0.62 0.50 Val GTA 0.00 0.05 0.25 0.03 0.09 0.07 0.09 0.02Val GTT 0.11 0.14 0.24 0.09 0.14 0.17 0.09 0.06 Val GTC 0.18 0.27 0.140.28 0.23 0.30 0.21 0.42 Cylindro- Amphi- Symbiodinium Amino DunaliellaDunaliella Gonium Phaeodactylum Chaetoceros theca dinium micro- AcidCodon viridis salina pectorale tricornutum compressum fusiformiscarterae adriacticum Ala GCG 0.13 0.15 0.43 0.15 0.08 0.07 0.17 0.22 AlaGCA 0.27 0.20 0.09 0.10 0.37 0.14 0.33 0.26 Ala GCT 0.25 0.27 0.08 0.230.36 0.35 0.29 0.20 Ala GCC 0.35 0.39 0.41 0.52 0.18 0.43 0.20 0.32 ArgAGG 0.25 0.22 0.13 0.02 0.14 0.09 0.15 0.27 Arg AGA 0.06 0.05 0.00 0.040.29 0.14 0.03 0.27 Arg CGG 0.08 0.12 0.40 0.10 0.00 0.06 0.08 0.09 ArgCGA 0.06 0.06 0.05 0.12 0.19 0.16 0.18 0.09 Arg CGT 0.15 0.13 0.08 0.410.38 0.34 0.18 0.09 Arg CGC 0.39 0.43 0.35 0.31 0.00 0.22 0.40 0.18 AsnAAT 0.17 0.23 0.07 0.30 0.58 0.42 0.37 0.21 Asn AAC 0.83 0.77 0.93 0.650.42 0.58 0.63 0.79 Asp GAT 0.38 0.40 0.11 0.41 0.53 0.54 0.54 0.50 AspGAC 0.62 0.60 0.89 0.59 0.47 0.46 0.46 0.50 Cys TGT 0.24 0.17 0.20 0.390.44 0.44 0.75 0.50 Cys TGC 0.76 0.83 0.90 0.61 0.56 0.56 0.25 0.50 EndTGA 0.31 0.37 0.50 0.06 0.50 0.13 0.50 1.00 End TAG 0.15 0.14 0.00 0.130.00 0.10 0.00 0.00 End TAA 0.54 0.49 0.50 0.81 0.50 0.77 0.50 0.00 GlnCAG 0.36 0.32 0.31 0.23 0.16 0.12 0.33 0.28 Gln CAA 0.12 0.08 0.07 0.140.19 0.25 0.15 0.17 Glu GAG 0.44 0.51 0.56 0.21 0.28 0.23 0.41 0.50 GluGAA 0.09 0.09 0.07 0.42 0.37 0.39 0.10 0.06 Gly GGG 0.14 0.10 0.18 0.080.12 0.06 0.19 0.32 Gly GGA 0.11 0.12 0.09 0.34 0.33 0.47 0.10 0.12 GlyGGT 0.22 0.22 0.07 0.30 0.39 0.35 0.34 0.16 Gly GGC 0.54 0.56 0.65 0.280.16 0.12 0.37 0.40 His CAT 0.25 0.25 0.43 0.28 0.84 0.39 0.12 0.40 HisCAC 0.75 0.75 0.57 0.72 0.16 0.61 0.88 0.60 Ile ATA 0.03 0.03 0.07 0.030.12 0.06 0.05 0.00 Ile ATT 0.25 0.31 0.33 0.51 0.65 0.42 0.53 0.38 IleATC 0.72 0.66 0.59 0.46 0.23 0.52 0.42 0.63 Leu TTG 0.11 0.12 0.04 0.260.11 0.26 0.35 0.39 Leu TTA 0.01 0.01 0.00 0.02 0.14 0.09 0.01 0.00 LeuCTG 0.60 0.61 0.64 0.15 0.05 0.09 0.22 0.39 Leu CTA 0.05 0.04 0.01 0.050.08 0.05 0.00 0.04 Leu CTT 0.07 0.08 0.05 0.18 0.51 0.37 0.31 0.13 LeuCTC 0.16 0.14 0.26 0.34 0.11 0.13 0.12 0.04 Lys AAG 0.87 0.89 0.93 0.750.52 0.60 0.93 0.85 Lys AAA 0.13 0.11 0.07 0.25 0.48 0.40 0.07 0.15 MetATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Phe TTT 0.25 0.29 0.10 0.440.65 0.37 0.21 0.25 Phe TTC 0.75 0.71 0.90 0.56 0.35 0.63 0.79 0.75 ProCCG 0.10 0.08 0.53 0.29 0.05 0.11 0.14 0.18 Pro CCA 0.10 0.17 0.09 0.120.45 0.33 0.42 0.09 Pro CCT 0.10 0.30 0.04 0.20 0.33 0.32 0.22 0.41 ProCCC 0.10 0.45 0.34 0.40 0.17 0.24 0.22 0.32 Ser AGT 0.09 0.06 0.02 0.120.14 0.12 0.13 0.09 Ser AGC 0.31 0.32 0.20 0.12 0.07 0.09 0.24 0.14 SerTCG 0.04 0.06 0.42 0.19 0.08 0.13 0.03 0.05 Ser TCA 0.08 0.10 0.09 0.060.31 0.12 0.25 0.05 Ser TCT 0.17 0.15 0.07 0.15 0.23 0.30 0.16 0.23 SerTCC 0.31 0.30 0.20 0.35 0.18 0.24 0.19 0.45 Thr ACG 0.16 0.13 0.42 0.230.10 0.09 0.14 0.10 Thr ACA 0.21 0.18 0.03 0.13 0.38 0.15 0.28 0.10 ThrACT 0.18 0.23 0.08 0.19 0.27 0.39 0.12 0.10 Thr ACC 0.46 0.46 0.47 0.450.25 0.37 0.47 0.70 Trp TGG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 TyrTAT 0.16 0.21 0.12 0.18 0.67 0.38 0.32 0.20 Tyr TAC 0.84 0.79 0.88 0.820.33 0.62 0.68 0.80 Val GTG 0.64 0.62 0.57 0.22 0.30 0.11 0.65 0.67 ValGTA 0.03 0.05 0.04 0.09 0.27 0.06 0.05 0.00 Val GTT 0.11 0.11 0.04 0.220.10 0.38 0.08 0.11 Val GTC 0.22 0.23 0.35 0.47 0.33 0.07 0.17 0.22Nanno- Amino chlorop- Cyclotella Navicula Thalassiosira C. YarrowiaMortierella Rhodococcus Acid Codon sis sp cryptica pelliculosapseudonana reinhardtii lipolytica alpina opacus Ala GCG 0.24 0.11 0.000.11 0.35 0.08 0.14 0.35 Ala GCA 0.10 0.16 0.13 0.25 0.08 0.11 0.12 0.14Ala GCT 0.17 0.45 0.44 0.33 0.13 0.35 0.29 0.09 Ala GCC 0.48 0.27 0.440.30 0.43 0.46 0.45 0.43 Arg AGG 0.00 0.09 0.05 0.18 0.05 0.05 0.05 0.05Arg AGA 0.00 0.05 0.10 0.17 0.01 0.13 0.06 0.02 Arg CGG 0.00 0.04 0.050.06 0.20 0.12 0.06 0.26 Arg CGA 0.29 0.08 0.35 0.11 0.04 0.52 0.09 0.12Arg CGT 0.14 0.47 0.20 0.34 0.09 0.11 0.32 0.11 Arg CGC 0.57 0.28 0.250.15 0.62 0.07 0.42 0.44 Asn AAT 0.00 0.25 0.47 0.43 0.09 0.17 0.15 0.21Asn AAC 1.00 0.75 0.53 0.57 0.91 0.83 0.85 0.79 Asp GAT 0.20 0.52 0.200.56 0.14 0.35 0.42 0.24 Asp GAC 0.80 0.48 0.80 0.44 0.86 0.65 0.58 0.76Cys TGT 0.00 0.29 0.10 0.54 0.10 0.46 0.13 0.26 Cys TGC 1.00 0.71 0.900.46 0.90 0.54 0.87 0.74 End TGA 0.00 0.10 0.00 0.31 0.27 0.16 0.05 0.72End TAG 0.00 0.00 0.00 0.38 0.22 0.38 0.25 0.17 End TAA 1.00 0.90 1.000.31 0.52 0.46 0.70 0.11 Gln CAG 0.41 0.19 0.21 0.16 0.38 0.33 0.36 0.28Gln CAA 0.00 0.17 0.28 0.19 0.04 0.08 0.06 0.06 Glu GAG 0.59 0.38 0.170.40 0.55 0.44 0.49 0.45 Glu GAA 0.00 0.26 0.34 0.26 0.03 0.14 0.09 0.22Gly GGG 0.10 0.10 0.03 0.12 0.11 0.05 0.03 0.18 Gly GGA 0.05 0.45 0.280.51 0.06 0.28 0.29 0.15 Gly GGT 0.25 0.22 0.13 0.23 0.11 0.32 0.32 0.20Gly GGC 0.60 0.24 0.56 0.14 0.72 0.34 0.36 0.48 His CAT 0.00 0.42 1.000.50 0.11 0.34 0.27 0.20 His CAC 1.00 0.58 0.00 0.50 0.89 0.66 0.73 0.80Ile ATA 0.00 0.04 0.00 0.08 0.03 0.03 0.01 0.05 Ile ATT 0.14 0.53 0.730.38 0.22 0.44 0.33 0.14 Ile ATC 0.86 0.42 0.27 0.54 0.75 0.53 0.66 0.81Leu TTG 0.22 0.20 0.16 0.29 0.04 0.09 0.27 0.09 Leu TTA 0.00 0.03 0.000.05 0.01 0.02 0.00 0.01 Leu CTG 0.09 0.06 0.12 0.08 0.73 0.37 0.26 0.41Leu CTA 0.00 0.03 0.04 0.06 0.03 0.05 0.02 0.03 Leu CTT 0.04 0.39 0.360.20 0.05 0.18 0.12 0.06 Leu CTC 0.65 0.29 0.32 0.32 0.15 0.29 0.32 0.40Lys AAG 1.00 0.70 0.83 0.76 0.95 0.84 0.91 0.80 Lys AAA 0.00 0.30 0.170.24 0.05 0.16 0.09 0.20 Met ATG 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00Phe TTT 0.20 0.31 0.78 0.38 0.16 0.38 0.39 0.09 Phe TTC 0.80 0.69 0.220.62 0.84 0.62 0.61 0.91 Pro CCG 0.08 0.10 0.21 0.16 0.33 0.10 0.07 0.52Pro CCA 0.08 0.16 0.29 0.31 0.08 0.10 0.08 0.09 Pro CCT 0.25 0.35 0.210.31 0.13 0.32 0.36 0.07 Pro CCC 0.58 0.39 0.29 0.23 0.47 0.47 0.49 0.32Ser AGT 0.00 0.09 0.13 0.18 0.04 0.07 0.05 0.08 Ser AGC 0.13 0.08 0.280.11 0.35 0.11 0.14 0.23 Ser TCG 0.00 0.15 0.25 0.17 0.25 0.16 0.32 0.33Ser TCA 0.00 0.12 0.08 0.12 0.05 0.08 0.08 0.07 Ser TCT 0.13 0.39 0.250.23 0.07 0.28 0.12 0.05 Ser TCC 0.75 0.18 0.03 0.19 0.25 0.30 0.29 0.24Thr ACG 0.28 0.10 0.18 0.21 0.30 0.11 0.17 0.28 Thr ACA 0.00 0.15 0.090.19 0.08 0.14 0.10 0.11 Thr ACT 0.17 0.33 0.41 0.28 0.10 0.26 0.23 0.07Thr ACC 0.56 0.43 0.32 0.32 0.52 0.49 0.49 0.53 Trp TGG 1.00 1.00 1.001.00 1.00 1.00 1.00 1.00 Tyr TAT 0.00 0.38 0.20 0.39 0.10 0.18 0.20 0.18Tyr TAC 1.00 0.62 0.80 0.61 0.90 0.82 0.80 0.82 Val GTG 0.31 0.16 0.180.29 0.67 0.33 0.22 0.37 Val GTA 0.00 0.09 0.09 0.16 0.03 0.05 0.02 0.05Val GTT 0.15 0.42 0.09 0.28 0.07 0.26 0.27 0.10 Val GTC 0.54 0.33 0.640.27 0.22 0.36 0.49 0.49

TABLE 20 Lipid biosynthesis pathway proteins. 3-Ketoacyl ACP synthaseCuphea hookeriana 3-ketoacyl-ACP synthase (GenBank Acc. No. AAC68861.1),Cuphea wrightii beta-ketoacyl-ACP synthase II (GenBank Acc. No.AAB37271.1), Cuphea lanceolata beta-ketoacyl-ACP synthase IV (GenBankAcc. No. CAC59946.1), Cuphea wrightii beta-ketoacyl-ACP synthase II(GenBank Acc. No. AAB37270.1), Ricinus communis ketoacyl-ACP synthase(GenBank Acc. No. XP_002516228), Gossypium hirsutum ketoacyl-ACPsynthase (GenBank Acc. No. ADK23940.1), Glycine max plastid3-keto-acyl-ACP synthase II-A (GenBank Acc No. AAW88763.1), Elaeisguineensis beta- ketoacyl-ACP synthase II (GenBank Acc. No. AAF26738.2),Helianthus annuus plastid 3- keto-acyl-ACP synthase I (GenkBank Acc. No.ABM53471.1), Glycine max 3-keto-acyl- ACP synthase I (GenkBank Acc. No.NP_001238610.1), Helianthus annuus plastid 3- keto-acyl-ACP synthase II(GenBank Acc ABI18155.1), Brassica napus beta-ketoacyl- ACP synthetase 2(GenBank Acc. No. AAF61739.1), Perilla frutescens beta-ketoacyl- ACPsynthase II (GenBank Acc. No. AAC04692.1), Helianthus annusbeta-ketoacyl-ACP synthase II (GenBank Accession No. ABI18155), Ricinuscommunis beta-ketoacyl-ACP synthase II (GenBank Accession No. AAA33872),Haematococcus pluvialis beta-ketoacyl acyl carrier protein synthase(GenBank Accession No. HM560033.1), Jatropha curcasbeta ketoacyl-ACPsynthase I (GenBank Accession No. ABJ90468.1), Populus trichocarpabeta-ketoacyl-ACP synthase I (GenBank Accession No. XP_002303661.1),Coriandrum sativum beta-ketoacyl-ACP synthetase I (GenBank Accession No.AAK58535.1), Arabidopsis thaliana 3-oxoacyl-[acyl-carrier-protein]synthase I (GenBank Accession No. NP_001190479.1), Vitis vinifera3-oxoacyl-[acyl-carrier-protein] synthase I (GenBank Accession No.XP_002272874.2) Fatty acyl-ACP Thioesterases Umbellularia californicafatty acyl-ACP thioesterase (GenBank Acc. No. AAC49001), Cinnamomumcamphora fatty acyl-ACP thioesterase (GenBank Acc. No. Q39473),Umbellularia californica fatty acyl-ACP thioesterase (GenBank Acc. No.Q41635), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc.No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBankAcc. No. AAB71730), Elaeis guineensis fatty acyl-ACP thioesterase(GenBank Acc. No. ABD83939), Elaeis guineensis fatty acyl-ACPthioesterase (GenBank Acc. No. AAD42220), Populus tomentosa fattyacyl-ACP thioesterase (GenBank Acc. No. ABC47311), Arabidopsis thalianafatty acyl- ACP thioesterase (GenBank Acc. No. NP_172327), Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank Acc. No. CAA85387),Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank Acc. No.CAA85388), Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc.No. Q9SQI3), Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank Acc.No. CAA54060), Cuphea hookeriana fatty acyl-ACP thioesterase (GenBankAcc. No. AAC72882), Cuphea calophylla subsp. mesostemon fatty acyl-ACPthioesterase (GenBank Acc. No. ABB71581), Cuphea lanceolata fattyacyl-ACP thioesterase (GenBank Acc. No. CAC19933), Elaeis guineensisfatty acyl-ACP thioesterase (GenBank Acc. No. AAL15645), Cupheahookeriana fatty acyl-ACP thioesterase (GenBank Acc. No. Q39513),Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank Acc. No.AAD01982), Vitis vinifera fatty acyl-ACP thioesterase (GenBank Acc. No.CAN81819), Garcinia mangostana fatty acyl-ACP thioesterase (GenBank Acc.No. AAB51525), Brassica juncea fatty acyl-ACP thioesterase (GenBank Acc.No. ABI18986), Madhuca longifolia fatty acyl-ACP thioesterase (GenBankAcc. No. AAX51637), Brassica napus fatty acyl-ACP thioesterase (GenBankAcc. No. ABH11710), Brassica napus fatty acyl-ACP thioesterase (GenBankAcc. No. CAA52070.1), Oryza sativa (indica cultivar-group) fattyacyl-ACP thioesterase (GenBank Acc. No. EAY86877), Oryza sativa(japonica cultivar-group) fatty acyl-ACP thioesterase (GenBank Acc. No.NP_001068400), Oryza sativa (indica cultivar-group) fatty acyl-ACPthioesterase (GenBank Acc. No. EAY99617), Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank Acc. No. AAC49269), Ulmus Americana fattyacyl-ACP thioesterase (GenBank Acc. No. AAB71731), Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank Acc. No. CAB60830), Cupheapalustris fatty acyl-ACP thioesterase (GenBank Acc. No. AAC49180), Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858, Irisgermanica fatty acyl-ACP thioesterase (GenBank Acc. No. AAG43858.1),Cuphea palustris fatty acyl-ACP thioesterase (GenBank Acc. No.AAC49179), Myristica fragrans fatty acyl-ACP thioesterase (GenBank Acc.No. AAB71729), Myristica fragrans fatty acyl-ACP thioesterase (GenBankAcc. No. AAB717291.1), Cuphea hookeriana fatty acyl-ACP thioesteraseGenBank Acc. No. U39834), Umbelluaria californica fatty acyl-ACPthioesterase (GenBank Acc. No. M94159), Cinnamomum camphora fattyacyl-ACP thioesterase (GenBank Acc. No. U31813), Ricinus communis fattyacyl-ACP thioesterase (GenBank Acc. No. ABS30422.1), Helianthus annuusacyl-ACP thioesterase (GenBank Accession No. AAL79361.1), Jatrophacurcas acyl-ACP thioesterase (GenBank Accession No. ABX82799.3), Zeamays oleoyl-acyl carrier protein thioesterase, (GenBank Accession No.ACG40089.1), Haematococcus pluvialis fatty acyl-ACP thioesterase(GenBank Accession No. HM560034.1) Desaturase Enzymes Linumusitatissimum fatty acid desaturase 3C, (GenBank Acc. No. ADV92272.1),Ricinus communis omega-3 fatty acid desaturase, endoplasmic reticulum,putative, (GenBank Acc. No. EEF36775.1), Vemicia fordii omega-3 fattyacid desaturase, (GenBank Acc. No. AAF12821), Glycine max chloroplastomega 3 fatty acid desaturase isoform 2, (GenBank Acc. No. ACF19424.1),Prototheca moriformis FAD-D omega 3 desaturase (SEQ ID NO: 35),Prototheca moriformis linoleate desaturase (SEQ ID NO: 36), Carthamustinctorius delta 12 desaturase, (GenBank Accession No. ADM48790.1),Gossypium hirsutum omega-6 desaturase, (GenBank Accession No.CAA71199.1), Glycine max microsomal desaturase (GenBank Accession No.BAD89862.1), Zea mays fatty acid desaturase (GenBank Accession No.ABF50053.1), Brassica napa linoleic acid desaturase (GenBank AccessionNo. AAA32994.1), Camelina sativa omega-3 desaturase (SEQ ID NO: 37),Prototheca moriformis delta 12 desaturase allele 2 (SEQ ID NO: 38,Camelina sativa omega-3 FAD7-1 (SEQ ID NO: 39), Helianthus annuusstearoyl-ACP desaturase, (GenBank Accession No. AAB65145.1), Ricinuscommunis stearoyl-ACP desaturase, (GenBank Accession No. AACG59946.1),Brassica juncea plastidic delta-9-stearoyl-ACP desaturase (GenBankAccession No. AAD40245.1), Glycine max stearoyl-ACP desaturase (GenBankAccession No. ACJ39209.1), Olea europaea stearoyl-ACP desaturase(GenBank Accession No. AAB67840.1), Vernicia fordiistearoyl-acyl-carrier protein desaturase, (GenBank Accession No.ADC32803.1), Descurainia sophia delta-12 fatty acid desaturase (GenBankAccession No. ABS86964.2), Euphorbia lagascae delta12-oleic aciddesaturase (GenBank Acc. No. AAS57577.1), Chlorella vulgaris delta 12fatty acid desaturease (GenBank Accession No. ACF98528), Chlorellavulgaris omega-3 fatty acid desaturease (GenBank Accession No.BAB78717), Haematococcus pluvialis omega-3 fatty acid desaturase(GenBank Accession No. HM560035.1), Haematococcus pluvialisstearoyl-ACP-desaturase GenBank Accession No. EF586860.1, Haematococcuspluvialis stearoyl-ACP-desaturase GenBank Accession No. EF523479.1Oleate 12-hydroxylase Enzymes Ricinus communis oleate 12-hydroxylase(GenBank Acc. No. AAC49010.1), Physaria lindheimeri oleate12-hydroxylase (GenBank Acc. No. ABQ01458.1), Physaria lindheimerimutant bifunctional oleate 12-hydroxylase:desaturase (GenBank Acc. No.ACF17571.1), Physaria lindheimeri bifunctional oleate 12-hydroxylase:desaturase (GenBank Accession No. ACQ42234.1), Physarialindheimeri bifunctional oleate 12-hydroxylase:desaturase (GenBank Acc.No. AAC32755.1), Arabidopsis lyrata subsp. Lyrata (GenBank Acc. No.XP_002884883.1) Glycerol-3-phosphate Enzymes Arabidopsis thalianaglycerol-3-phosphate acyltransferase BAA00575, Chlamydomonas reinhardtiiglycerol-3-phosphate acyltransferase (GenBank Acc. No. EDP02129),Chlamydomonas reinhardtii glycerol-3-phosphate acyltransferase (GenBankAcc. No. Q886Q7), Cucurbita moschataacyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase(GenBank Acc. No. BAB39688), Elaeis guineensis glycerol-3-phosphateacyltransferase, ((GenBank Acc. No. AAF64066), Garcina mangostanaglycerol-3- phosphate acyltransferase (GenBank Acc. No. ABS86942),Gossypium hirsutum glycerol- 3-phosphate acyltransferase (GenBank Acc.No. ADK23938), Jatropha curcas glycerol-3- phosphate acyltransferase(GenBank Acc. No. ADV77219), Jatropha curcas plastidglycerol-3-phosphate acyltransferase (GenBank Acc. No. ACR61638),Ricinus communis plastidial glycerol-phosphate acyltransferase (GenBankAcc. No. EEF43526), Vica faba glycerol-3-phosphate acyltransferase(GenBank Accession No. AAD05164), Zea mays glycerol-3-phosphateacyltransferase (GenBank Acc. No. ACG45812) Lysophosphatidic acidacyltransferase Enzymes Arabidopsis thaliana1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.AEE85783), Brassica juncea 1-acyl-sn-glycerol-3-phosphateacyltransferase (GenBank Accession No. ABQ42862), Brassica juncea1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.ABM92334), Brassica napus 1-acyl-sn-glycerol- 3-phosphateacyltransferase (GenBank Accession No. CAB09138), Chlamydomonasreinhardtii lysophosphatidic acid acyltransferase (GenBank Accession No.EDP02300), Cocos nucifera lysophosphatidic acid acyltransferase (GenBankAcc. No. AAC49119), Limnanthes alba lysophosphatidic acidacyltransferase (GenBank Accession No. EDP02300), Limnanthes douglasii1-acyl-sn-glycerol-3-phosphate acyltransferase (putative) (GenBankAccession No. CAA88620), Limnanthes douglasii acyl-CoA:sn-1-acylglycerol-3-phosphate acyltransferase (GenBank Accession No.ABD62751), Limnanthes douglasii 1-acylglycerol-3-phosphateO-acyltransferase (GenBank Accession No. CAA58239), Ricinus communis1-acyl-sn-glycerol-3-phosphate acyltransferase (GenBank Accession No.EEF39377) Diacylglycerol acyltransferase Enzymes Arabidopsis thalianadiacylglycerol acyltransferase (GenBank Acc. No. CAB45373), Brassicajuncea diacylglycerol acyltransferase (GenBank Acc. No. AAY40784),Elaeis guineensis putative diacylglycerol acyltransferase (GenBank Acc.No. AEQ94187), Elaeis guineensis putative diacylglycerol acyltransferase(GenBank Acc. No. AEQ94186), Glycine max acyl CoA:diacylglycerolacyltransferase (GenBank Acc. No. AAT73629), Helianthus annusdiacylglycerol acyltransferase (GenBank Acc. No. ABX61081), Oleaeuropaea acyl- CoA:diacylglycerol acyltransferase 1 (GenBank Acc. No.AAS01606), Ricinus communis diacylglycerol acyltransferase (GenBank Acc.No. AAR11479) Phospholipid diacylglycerol acyltransferase EnzymesArabidopsis thaliana phospholipid:diacylglycerol acyltransferase(GenBank Acc. No. AED91921), Elaeis guineensis putativephospholipid:diacylglycerol acyltransferase (GenBank Acc. No. AEQ94116),Glycine max phospholipid:diacylglycerol acyltransferase 1-like (GenBankAcc. No. XP_003541296), Jatropha curcas phospholipid:diacylglycerolacyltransferase (GenBank Acc. No. AEZ56255), Ricinus communisphospholipid:diacylglycerol acyltransferase (GenBank Acc. No. ADK92410),Ricinus communis phospholipid:diacylglycerol acyltransferase (GenBankAcc. No. AEW99982)

Example 10 Engineering Chlorella sorokiniana

Expression of recombinant genes in accordance with the present inventionin Chlorella sorokiniana can be accomplished by modifying the methodsand vectors taught by Dawson et al. as discussed herein. Briefly, Dawsonet al., Current Microbiology Vol. (1997) pp. 356-362, reported thestable nuclear transformation of Chlorella sorokiniana with plasmid DNA.Using the transformation method of microprojectile bombardment, Dawsonintroduced the plasmid pSV72-NR9, encoding the full Chlorella vulgarisnitrate reductase gene (NR, GenBank Accession No. U39931), into mutantChlorella sorokiniana (NR-mutants). The NR-mutants are incapable ofgrowth without the use of nitrate as a source of nitrogen. Nitratereductase catalyzes the conversion of nitrate to nitrite. Prior totransformation, Chlorella sorokiniana NR-mutants were unable to growbeyond the microcolony stage on culture medium comprising nitrate (NO₃⁻) as the sole nitrogen source. The expression of the Chlorella vulgarisNR gene product in NR-mutant Chlorella sorokiniana was used as aselectable marker to rescue the nitrate metabolism deficiency. Upontransformation with the pSV72-NR9 plasmid, NR-mutant Chlorellasorokiniana stably expressing the Chlorella vulgaris NR gene productwere obtained that were able to grow beyond the microcolony stage onagar plates comprising nitrate as the sole carbon source. Evaluation ofthe DNA of the stable transformants was performed by Southern analysisand evaluation of the RNA of the stable transformants was performed byRNase protection. Selection and maintenance of the transformed Chlorellasorokiniana (NR mutant) was performed on agar plates (pH 7.4) comprising0.2 g/L MgSO₄, 0.67 g/L KH₂PO₄, 3.5 g/L K₂HPO₄, 1.0 g/L Na₃C₆H₅O₇.H₂Oand 16.0 g/L agar, an appropriate nitrogen source (e.g., NO₃ ⁻),micronutrients, and a carbon source. Dawson also reported thepropagation of Chlorella sorokiniana and Chlorella sorokiniana NRmutants in liquid culture medium. Dawson reported that the plasmidpSV72-NR9 and the promoter and 3′ UTR/terminator of the Chlorellavulgaris nitrate reductase gene were suitable to enable heterologousgene expression in Chlorella sorokiniana NR-mutants. Dawson alsoreported that expression of the Chlorella vulgaris nitrate reductasegene product was suitable for use as a selectable marker in Chlorellasorokiniana NR-mutants.

In an embodiment of the present invention, vector pSV72-NR9, comprisingnucleotide sequence encoding the Chlorella vulgaris nitrate reductase(CvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellasorokiniana to reflect the codon bias inherent in nuclear genes ofChlorella sorokiniana in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the CvNR promoterupstream of the protein-coding sequence and operably linked to the CvNR3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chlorella sorokiniana genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorellasorokiniana with the transformation vector is achieved throughwell-known transformation techniques including microprojectilebombardment or other known methods. Activity of the CvNR gene productcan be used as a selectable marker to rescue the nitrogen assimilationdeficiency of Chlorella sorokiniana NR mutant strains and to select forChlorella sorokiniana NR-mutants stably expressing the transformationvector. Growth media suitable for Chlorella sorokiniana lipid productioninclude, but are not limited to 0.5 g/L KH₂PO₄, 0.5 g/L K₂HPO₄, 0.25 g/LMgSO₄.7H₂O, with supplemental micronutrients and the appropriatenitrogen and carbon sources (Patterson, Lipids Vol. 5:7 (1970), pp.597-600). Evaluation of fatty acid profiles of Chlorella sorokinianalipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 11 Engineering Chlorella vulgaris

Expression of recombinant genes in accordance with the present inventionin Chlorella vulgaris can be accomplished by modifying the methods andvectors taught by Chow and Tung et al. as discussed herein. Briefly,Chow and Tung et al., Plant Cell Reports, Volume 18 (1999), pp. 778-780,reported the stable nuclear transformation of Chlorella vulgaris withplasmid DNA. Using the transformation method of electroporation, Chowand Tung introduced the plasmid pIG121-Hm (GenBank Accession No.AB489142) into Chlorella vulgaris. The nucleotide sequence of pIG121-Hmcomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter upstream of the GUSprotein-coding sequence and further operably linked to the 3′UTR/terminator of the nopaline synthase (nos) gene downstream of the GUSprotein-coding sequence. The sequence of plasmid pIG121-Hm furthercomprised a hygromycin B antibiotic resistance cassette. This hygromycinB antibiotic resistance cassette comprised a CaMV 35S promoter operablylinked to sequence encoding the hygromycin phosphotransferase (hpt,GenBank Accession No. BAH24259) gene product. Prior to transformation,Chlorella vulgaris was unable to be propagated in culture mediumcomprising 50 ug/ml hygromycin B. Upon transformation with the pIG121-Hmplasmid, transformants of Chlorella vulgaris were obtained that werepropagated in culture medium comprising 50 ug/ml hygromycin B. Theexpression of the hpt gene product in Chlorella vulgaris enabledpropagation of transformed Chlorella vulgaris in the presence of 50ug/mL hygromycin B, thereby establishing the utility of the a hygromycinB resistance cassette as a selectable marker for use in Chlorellavulgaris. Detectable activity of the GUS reporter gene indicated thatCaMV 35S promoter and nos 3′UTR are suitable for enabling heterologousgene expression in Chlorella vulgaris. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Selectionand maintenance of transformed Chlorella vulgaris was performed on agarplates comprising YA medium (agar and 4 g/L yeast extract). Thepropagation of Chlorella vulgaris in liquid culture medium was conductedas discussed by Chow and Tung. Propagation of Chlorella vulgaris inmedia other than YA medium has been described (for examples, see Chaderet al., Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26 andIllman et al., Enzyme and Microbial Technology, Vol. 27 (2000), pp.631-635). Chow and Tung reported that the plasmid pIG121-Hm, the CaMV35S promoter, and the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator are suitable to enable heterologous gene expression inChlorella vulgaris. In addition, Chow and Tung reported the hygromycin Bresistance cassette was suitable for use as a selectable marker inChlorella vulgaris. Additional plasmids, promoters, 3′UTR/terminators,and selectable markers suitable for enabling heterologous geneexpression in Chlorella vulgaris have been discussed in Chader et al.,Revue des Energies Renouvelabes, Volume 14 (2011), pp. 21-26.

In an embodiment of the present invention, pIG121-Hm, comprising thenucleotide sequence encoding the hygromycin B gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Chlorella vulgaris to reflect thecodon bias inherent in nuclear genes of Chlorella vulgaris in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the CaMV 35S promoter upstream of the protein-coding sequenceand operably linked to the Agrobacterium tumefaciens nopaline synthasegene 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Chlorella vulgaris genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chlorella vulgariswith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the hygromycin B resistance gene product can beused as a marker to select for Chlorella vulgaris transformed with thetransformation vector on, but not limited to, agar medium comprisinghygromycin. Growth media suitable for Chlorella vulgaris lipidproduction include, but are not limited to BG11 medium (0.04 g/L KH₂PO₄,0.075 g/L CaCl₂, 0.036 g/L citric acid, 0.006 g/L Ammonium FerricCitrate, 1 mg/L EDTA, and 0.02 g/L Na₂CO₃) supplemented with tracemetals, and optionally 1.5 g/L NaNO3. Additional media suitable forculturing Chlorella vulgaris for lipid production include, for example,Watanabe medium (comprising 1.5 g/L KNO₃, 1.25 g/L KH₂PO₄, 1.25 gl⁻¹MgSO₄.7H₂O, 20 mgl⁻¹ FeSO₄.7H₂O with micronutrients and low-nitrogenmedium (comprising 203 mg/l (NH₄)₂HPO₄, 2.236 g/l KCl, 2.465 g/l MgSO₄,1.361 g/l KH₂PO₄ and 10 mg/l FeSO₄) as reported by Illman et al., Enzymeand Microbial Technology, Vol. 27 (2000), pp. 631-635. Evaluation offatty acid profiles of Chlorella vulgaris lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 12 Engineering Chlorella ellipsoidea

Expression of recombinant genes in accordance with the present inventionin Chlorella ellipsoidea can be accomplished by modifying the methodsand vectors taught by Chen et al. as discussed herein. Briefly, Chen etal., Current Genetics, Vol. 39:5 (2001), pp. 365-370, reported thestable transformation of Chlorella ellipsoidea with plasmid DNA. Usingthe transformation method of electroporation, Chen introduced theplasmid pBinUΩNP-1 into Chlorella ellipsoidea. The nucleotide sequenceof pBinUΩNP-1 comprised sequence encoding the neutrophil peptide-1(NP-1) rabbit gene product operably linked to a Zea mays Ubiquitin(ubi1) gene promoter upstream of the NP-1 protein-coding region andoperably linked to the 3′ UTR/terminator of the nopaline synthase (nos)gene downstream of the NP-1 protein-coding region. The sequence ofplasmid pBinUΩNP-1 further comprised a G418 antibiotic resistancecassette. This G418 antibiotic resistance cassette comprised sequenceencoding the aminoglycoside 3′-phosphotransferase (aph 3′) gene product.The aph 3′ gene product confers resistance to the antibiotic G418. Priorto transformation, Chlorella ellipsoidea was unable to be propagated inculture medium comprising 30 ug/mL G418. Upon transformation with thepBinUΩNP-1 plasmid, transformants of Chlorella ellipsoidea were obtainedthat were propagated in selective culture medium comprising 30 ug/mLG418. The expression of the aph 3′ gene product in Chlorella ellipsoideaenabled propagation of transformed Chlorella ellipsoidea in the presenceof 30 ug/mL G418, thereby establishing the utility of the G418antibiotic resistance cassette as selectable marker for use in Chlorellaellipsoidea. Detectable activity of the NP-1 gene product indicated thatthe ubi1 promoter and nos 3′ UTR are suitable for enabling heterologousgene expression in Chlorella ellipsoidea. Evaluation of the genomic DNAof the stable transformants was performed by Southern analysis.Selection and maintenance of the transformed Chlorella ellipsoidea wasperformed on Knop medium (comprising 0.2 g/L K₂HPO₄, 0.2 g/L MgSO₄.7H₂O,0.12 g/L KCl, and 10 mg/L FeCl3, pH 6.0-8.0 supplemented with 0.1% yeastextract and 0.2% glucose) with 15 ug/mL G418 (for liquid cultures) orwith 30 ug/mL G418 (for solid cultures comprising 1.8% agar).Propagation of Chlorella ellipsoidea in media other than Knop medium hasbeen reported (see Cho et al., Fisheries Science, Vol. 73:5 (2007), pp.1050-1056, Jarvis and Brown, Current Genetics, Vol. 19 (1991), pp.317-321 and Kim et al., Marine Biotechnology, Vol. 4 (2002), pp. 63-73).Additional plasmids, promoters, 3′UTR/terminators, and selectablemarkers suitable for enabling heterologous gene expression in Chlorellaellipsoidea have been reported (see Jarvis and Brown and Kim et al.,Marine Biotechnology, Vol. 4 (2002), pp. 63-73). Chen reported that theplasmid pBinUΩNP-1, the ubi1 promoter, and the Agrobacterium tumefaciensnopaline synthase gene 3′UTR/terminator are suitable to enable exogenousgene expression in Chlorella ellipsoidea. In addition, Chen reportedthat the G418 resistance cassette encoded on pBinUΩNP-1 was suitable foruse as a selectable marker in Chlorella ellipsoidea.

In an embodiment of the present invention, vector pBinUΩNP-1, comprisingthe nucleotide sequence encoding the aph 3′ gene product, conferringresistance to G418, for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellaellipsoidea to reflect the codon bias inherent in nuclear genes ofChlorella ellipsoidea in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Zea mays ubi1promoter upstream of the protein-coding sequence and operably linked tothe Agrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator atthe 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella ellipsoidea genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella ellipsoidea with the transformationvector is achieved through well-known transformation techniquesincluding electroporation or other known methods. Activity of the aph 3′gene product can be used as a marker to select for Chlorella ellipsoideatransformed with the transformation vector on, but not limited to, Knopagar medium comprising G418. Growth media suitable for Chlorellaellipsoidea lipid production include, but are not limited to, Knopmedium and those culture medium reported by Jarvis and Brown and Kim etal. Evaluation of fatty acid profiles of Chlorella ellipsoidea lipidscan be assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 13 Engineering Chlorella kessleri

Expression of recombinant genes in accordance with the present inventionin Chlorella kessleri can be accomplished by modifying the methods andvectors taught by El-Sheekh et al. as discussed herein. Briefly,El-Sheekh et al., Biologia Plantarium, Vol. 42:2 (1999), pp. 209-216,reported the stable transformation of Chlorella kessleri with plasmidDNA. Using the transformation method of microprojectile bombardment,El-Sheekh introduced the plasmid pBI121 (GenBank Accession No. AF485783)into Chlorella kessleri. Plasmid pBI121 comprised a kanamycin/neomycinantibiotic resistance cassette. This kanamycin/neomycin antibioticresistance cassette comprised the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter, sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) for resistance to kanamycin and G418, and the 3′UTR/terminator of the Agrobacterium tumefaciens nopaline synthase (nos)gene. pBI121 further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably linked to a CaMV 35S promoter andoperably linked to a 3′ UTR/terminator of the nos gene. Prior totransformation, Chlorella kessleri was unable to be propagated inculture medium comprising 15 ug/L kanamycin. Upon transformation withthe pBI121plasmid, transformants of Chlorella kessleri were obtainedthat were propagated in selective culture medium comprising 15 mg/Lkanamycin. The express ion of the nptII gene product in Chlorellakessleri enabled propagation in the presence of 15 mg/L kanamycin,thereby establishing the utility of the kanamycin/neomycin antibioticresistance cassette as selectable marker for use in Chlorella kessleri.Detectable activity of the GUS gene product indicated that the CaMV 35Spromoter and nos 3′ UTR are suitable for enabling heterologous geneexpression in Chlorella kessleri. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. As reported byEl-Sheekh, selection and maintenance of transformed Chlorella kessleriwas conducted on semisolid agar plates comprising YEG medium (1% yeastextract, 1% glucose) and 15 mg/L kanamycin. El-Sheekh also reported thepropagation of Chlorella kessleri in YEG liquid culture media.Additional media suitable for culturing Chlorella kessleri for lipidproduction are disclosed in Sato et al., BBA Molecular and Cell Biologyof Lipids, Vol. 1633 (2003), pp. 27-34). El-Sheekh reported that theplasmid pBI121, the CaMV promoter, and the nopaline synthase gene3′UTR/terminator are suitable to enable heterologous gene expression inChlorella kessleri. In addition, El-Sheekh reported that thekanamycin/neomycin resistance cassette encoded on pBI121 was suitablefor use as a selectable marker in Chlorella kessleri.

In an embodiment of the present invention, vector pBI121, comprising thenucleotide sequence encoding the kanamycin/neomycin resistance geneproduct for use as a selectable marker, is constructed and modified tofurther comprise a lipid biosynthesis pathway expression cassettesequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected from Table 20, eachprotein-coding sequence codon-optimized for expression in Chlorellakessleri to reflect the codon bias inherent in nuclear genes ofChlorella kessleri in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the CaMV 35S promoterupstream of the protein-coding sequence and operably linked to theAgrobacterium tumefaciens nopaline synthase gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Chlorella kessleri genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Chlorella kessleri with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nptII gene product can be used as a marker to select forChlorella kessleri transformed with the transformation vector on, butnot limited to, YEG agar medium comprising kanamycin or neomycin. Growthmedia suitable for Chlorella kessleri lipid production include, but arenot limited to, YEG medium, and those culture media reported by Sato etal. Evaluation of fatty acid profiles of Chlorella kessleri lipids canbe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 14 Engineering Dunaliella tertiolecta

Expression of recombinant genes in accordance with the present inventionin Dunaliella tertiolecta can be accomplished by modifying the methodsand vectors taught by Walker et al. as discussed herein. Briefly, Walkeret al., Journal of Applied Phycology, Vol. 17 (2005), pp. 363-368,reported stable nuclear transformation of Dunaliella tertiolecta withplasmid DNA. Using the transformation method of electroporation, Walkerintroduced the plasmid pDbleFLAG1.2 into Dunaliella tertiolecta.pDbleFLAG1.2 comprised sequence encoding a bleomycin antibioticresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotic phleomycin, operably linked to the promoter and 3′ UTR of theDunaliella tertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenasesmall subunit gene (rbcS1, GenBank Accession No. AY530155). Prior totransformation, Dunaliella tertiolecta was unable to be propagated inculture medium comprising 1 mg/L phleomycin. Upon transformation withthe pDbleFLAG1.2 plasmid, transformants of Dunaliella tertiolecta wereobtained that were propagated in selective culture medium comprising 1mg/L phleomycin. The expression of the ble gene product in Dunaliellatertiolecta enabled propagation in the presence of 1 mg/L phleomycin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Dunaliella tertiolecta.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. As reported by Walker, selection and maintenanceof transformed Dunaliella tertiolecta was conducted in Dunaliella medium(DM, as described by Provasoli et al., Archiv fur Mikrobiologie, Vol. 25(1957), pp. 392-428) further comprising 4.5 g/L NaCl and 1 mg/Lpheomycin. Additional media suitable for culturing Dunaliellatertiolecta for lipid production are discussed in Takagi et al., Journalof Bioscience and Bioengineering, Vol. 101:3 (2006), pp. 223-226 and inMassart and Hanston, Proceedings Venice 2010, Third InternationalSymposium on Energy from Biomass and Waste. Walker reported that theplasmid pDbleFLAG1.2 and the promoter and 3′ UTR of the Dunaliellatertiolecta ribulose-1,5-bisphosphate carboxylase/oxygenase smallsubunit gene are suitable to enable heterologous expression inDunaliella tertiolecta. In addition, Walker reported that the bleomycinresistance cassette encoded on pDbleFLAG1.2 was suitable for use as aselectable marker in Dunaliella tertiolecta.

In an embodiment of the present invention, vector pDbleFLAG1.2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Dunaliella tertiolecta to reflect thecodon bias inherent in nuclear genes of Dunaliella tertiolecta inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the rbcS1 promoter upstream of the protein-codingsequence and operably linked to the rbcS1 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella tertiolecta genome for targeted genomic integration ofthe transformation vector. Homology regions may be selected to disruptone or more genomic sites of endogenous lipid biosynthesis pathwaygenes. Stable transformation of Dunaliella tertiolecta with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product can be used as a marker to select for Dunaliellatertiolecta transformed with the transformation vector on, but notlimited to, DM medium comprising pheomycin. Growth medium suitable forDunaliella tertiolecta lipid production include, but are not limited toDM medium and those culture media described by Takagi et al. and Massartand Hanston. Evaluation of fatty acid profiles of Dunaliella tertiolectalipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 15 Engineering Volvox carteri

Expression of recombinant genes in accordance with the present inventionin Volvox carteri can be accomplished by modifying the methods andvectors taught by Hallman and Rappel et al. as discussed herein.Briefly, Hallman and Rappel et al., The Plant Journal, Volume 17 (1999),pp. 99-109, reported the stable nuclear transformation of Volvox carteriwith plasmid DNA. Using the transformation method of microprojectilebombardment, Hallman and Rappel introduced the pzeoE plasmid into Volvoxcarteri. The pzeoE plasmid comprised sequence encoding a bleomycinantibiotic resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein (ble), forresistance to the antibiotic zeocin, operably linked to and the promoterand 3′ UTR of the Volvox carteri beta-tubulin gene (GenBank AccessionNo. L24547). Prior to transformation, Volvox carteri was unable to bepropagated in culture medium comprising 1.5 ug/ml zeocin. Upontransformation with the pzeoE plasmid, transformants of Volvox carteriwere obtained that were propagated in selective culture mediumcomprising greater than 20 ug/ml zeocin. The expression of the ble geneproduct in Volvox carteri enabled propagation in the presence of 20ug/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use in Volvoxcarteri. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. As reported by Hallman and Rappel,selection and maintenance of transformed Volvox carteri was conducted inVolvox medium (VM, as described by Provasoli and Pintner, The Ecology ofAlgae, Special Publication No. 2 (1959), Tyron, C. A. and Hartman, R.T., eds., Pittsburgh: Univeristy of Pittsburgh, pp. 88-96) with 1 mg/Lpheomycin. Media suitable for culturing Volvox carteri for lipidproduction are also discussed by Starr in Starr R, C, Dev Biol Suppl.,Vol. 4 (1970), pp. 59-100). Hallman and Rappel reported that the plasmidpzeoE and the promoter and 3′ UTR of the Volvox carteri beta-tubulingene are suitable to enable heterologous expression in Volvox carteri.In addition, Hallman and Rappel reported that the bleomycin resistancecassette encoded on pzeoE was suitable for use as a selectable marker inVolvox carteri. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inVolvox carteri and suitable for use as selective markers Volvox carteriin have been reported (for instance see Hallamann and Sumper,Proceedings of the National Academy of Sciences, Vol. 91 (1994), pp11562-11566 and Hallman and Wodniok, Plant Cell Reports, Volume 25(2006), pp. 582-581).

In an embodiment of the present invention, vector pzeoE, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 19, each protein-coding sequencecodon-optimized for expression in Volvox carteri to reflect the codonbias inherent in nuclear genes of Volvox carteri in accordance withTables 19A-D. For each lipid biosynthesis pathway protein of Table 20,the codon-optimized gene sequence can individually be operably linked tothe Volvox carteri beta-tubulin promoter upstream of the protein-codingsequence and operably linked to the Volvox carteri beta-tubulin3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Volvox carteri genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Volvox carteri genome(referenced in the publication by Prochnik et al., Science, Vol.329:5988 (2010), pp 223-226). Stable transformation of Volvox carteriwith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the ble gene product can be used as a markerto select for Volvox carteri transformed with the transformation vectoron, but not limited to, VM medium comprising zeocin. Growth mediumsuitable for Volvox carteri lipid production include, but are notlimited to VM medium and those culture media discussed by Starr.Evaluation of fatty acid profiles of Volvox carteri lipids can beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 16 Engineering Haematococcus pluvialis

Expression of recombinant genes in accordance with the present inventionin Haematococcus pluvialis can be accomplished by modifying the methodsand vectors taught by Steinbrenner and Sandmann et al. as discussedherein. Briefly, Steinbrenner and Sandmann et al., Applied andEnvironmental Microbiology, Vol. 72:12 (2006), pp. 7477-7484, reportedthe stable nuclear transformation of Haematococcus pluvialis withplasmid DNA. Using the transformation method of microprojectilebombardment, Steinbrenner introduced the plasmid pPlat-pds-L504R intoHaematococcus pluvialis. The plasmid pPlat-pds-L504R comprised anorflurazon resistance cassette, which comprised the promoter,protein-coding sequence, and 3′UTR of the Haematococcus pluvialisphytoene desaturase gene (Pds, GenBank Accession No. AY781170), whereinthe protein-coding sequence of Pds was modified at position 504 (therebychanging a leucine to an arginine) to encode a gene product (Pds-L504R)that confers resistance to the herbicide norflurazon. Prior totransformation with pPlat-pds-L504R, Haematococcus pluvialis was unableto propagate on medium comprising 5 uM norflurazon. Upon transformationwith the pPlat-pds-L504R plasmid, transformants of Haematococcuspluvialis were obtained that were propagated in selective culture mediumcomprising 5 uM norflurazon. The expression of the Pds-L504R geneproduct in Haematococcus pluvialis enabled propagation in the presenceof 5 uM norflurazon, thereby establishing the utility of the norflurazonherbicide resistance cassette as selectable marker for use inHaematococcus pluvialis. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. As reported bySteinbrenner, selection and maintenance of transformed Haematococcuspluvialis was conducted on agar plates comprising OHA medium (OHM (0.41g/L KNO₃, 0.03 g/L Na₂HPO₄, 0.246 g/L MgSO₄.7H₂O, 0.11 g/L CaCl₂.2H₂O,2.62 mg/L Fe_((III))citrate×H₂O, 0.011 mg/L CoCl₂.6H₂O, 0.012 mg/LCuSO₄.5H₂O, 0.075 mg/L Cr₂O₃, 0.98 mg/L MnCl₂.4H₂O, 0.12 mg/LNa₂Moa₄×2H₂O, 0.005 mg/L SeO₂ and 25 mg/L biotin, 17.5 mg/L thiamine,and 15 mg/L vitamin B12), supplemented with 2.42 g/L Tris-acetate, and 5mM norflurazon. Propagation of Haematococcus pluvialis in liquid culturewas performed by Steinbrenner and Sandmann using basal medium (basalmedium as described by Kobayashi et al., Applied and EnvironmentalMicrobiology, Vol. 59 (1993), pp. 867-873). Steinbrenner and Sandmannreported that the pPlat-pds-L504R plasmid and promoter and 3′ UTR of theHaematococcus pluvialis phytoene desaturase gene are suitable to enableheterologous expression in Haematococcus pluvialis. In addition,Steinbrenner and Sandmann reported that the norflurazon resistancecassette encoded on pPlat-pds-L504R was suitable for use as a selectablemarker in Haematococcus pluvialis. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Haematococcus pluvialis have beenreported (see Kathiresan et al., Journal of Phycology, Vol. 45 (2009),pp 642-649).

In an embodiment of the present invention, vector pPlat-pds-L504R,comprising the nucleotide sequence encoding the Pds-L504R gene productfor use as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Haematococcus pluvialis to reflect thecodon bias inherent in nuclear genes of Haematococcus pluvialis inaccordance with Tables 19 A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Haematococcus pluvialis pds gene promoterupstream of the protein-coding sequence and operably linked to theHaematococcus pluvialis pds gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Haematococcuspluvialis genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Haematococcus pluvialis with the transformation vectoris achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thePds-L504R gene product can be used as a marker to select forHaematococcus pluvialis transformed with the transformation vector on,but not limited to, OHA medium comprising norflurazon. Growth mediasuitable for Haematococcus pluvialis lipid production include, but arenot limited to basal medium and those culture media described byKobayashi et al., Kathiresan et al, and Gong and Chen, Journal ofApplied Phycology, Vol. 9:5 (1997), pp. 437-444). Evaluation of fattyacid profiles of Haematococcus pluvialis lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 17 Engineering Closterium peracerosum-strigosum-littoralecomplex

Expression of recombinant genes in accordance with the present inventionin Closterium peracerosum-strigosum-littorale complex can beaccomplished by modifying the methods and vectors taught by Abe et al.as discussed herein. Briefly, Abe et al., Plant Cell Physiology, Vol.52:9 (2011), pp. 1676-1685, reported the stable nuclear transformationof Closterium peracerosum-strigosum-littorale complex with plasmid DNA.Using the transformation methods of microprojectile bombardment, Abeintroduced the plasmid pSA106 into Closteriumperacerosum-strigosum-littorale complex. Plasmid pSA106 comprised ableomycin resistance cassette, comprising sequence encoding theStreptoalloteichus hindustanus Bleomycin binding protein gene (ble,GenBank Accession No. CAA37050) operably linked to the promoter and 3′UTR of the Closterium peracerosum-strigosum-littorale complexChlorophyll a/b-binding protein gene (CAB, GenBank Accession No.AB363403). Prior to transformation with pSA106, Closteriumperacerosum-strigosum-littorale complex was unable to propagate onmedium comprising 3 ug/ml phleomycin. Upon transformation with pSA106,transformants of Closterium peracerosum-strigosum-littorale complex wereobtained that were propagated in selective culture medium comprising 3ug/ml phleomycin. The expression of the ble gene product in Closteriumperacerosum-strigosum-littorale complex enabled propagation in thepresence of 3 ug/ml phleomycin, thereby establishing the utility of thebleomycin antibiotic resistance cassette as selectable marker for use inClosterium peracerosum-strigosum-littorale complex. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. As reported by Abe, selection and maintenance of transformedClosterium peracerosum-strigosum-littorale complex was conducted firstin top agar with C medium (0.1 g/L KNO₃, 0.015 g/L Ca(NO₃)₂.4H2O, 0.05g/L glycerophosphate-Na2, 0.04 g/L MgSO₄.7H₂O, 0.5 g/L Tris(hydroxylmethyl) aminomethane, trace minerals, biotin, vitamins B₁ andB₁₂) and then subsequently isolated to agar plates comprising C mediumsupplemented with phleomycin. As reported by Abe, propagation ofClosterium peracerosum-strigosum-littorale complex in liquid culture wasperformed in C medium. Additional liquid culture medium suitable forpropagation of Closterium peracerosum-strigosum-littorale complex arediscussed by Sekimoto et al., DNA Research, 10:4 (2003), pp. 147-153.Abe reported that the pSA106 plasmid and promoter and 3′ UTR of theClosterium peracerosum-strigosum-littorale complex CAB gene are suitableto enable heterologous gene expression in Closteriumperacerosum-strigosum-littorale complex. In addition, Abe reported thatthe bleomycin resistance cassette encoded on pSA106 was suitable for useas a selectable marker in Closterium peracerosum-strigosum-littoralecomplex. Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inClosterium peracerosum-strigosum-littorale complex have been reported(see Abe et al., Plant Cell Physiology, Vol. 49 (2008), pp. 625-632).

In an embodiment of the present invention, vector pSA106, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Closteriumperacerosum-strigosum-littorale complex to reflect the codon biasinherent in nuclear genes of Closterium peracerosum-strigosum-littoralecomplex in accordance with Tables 19A-D. For each lipid biosynthesispathway protein of Table 20, the codon-optimized gene sequence canindividually be operably linked to the Closteriumperacerosum-strigosum-littorale complex CAB gene promoter upstream ofthe protein-coding sequence and operably linked to the Closteriumperacerosum-strigosum-littorale complex CAB gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Closterium peracerosum-strigosum-littorale complex genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofClosterium peracerosum-strigosum-littorale complex with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the ble gene product can be used as a marker to select forClosterium peracerosum-strigosum-littorale complex transformed with thetransformation vector on, but not limited to, C medium comprisingphleomycin. Growth media suitable for Closteriumperacerosum-strigosum-littorale complex lipid production include, butare not limited to C medium and those culture media reported by Abe etal. and Sekimoto et al. Evaluation of fatty acid profiles of Closteriumperacerosum-strigosum-littorale complex lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 18 Engineering Dunaliella viridis

Expression of recombinant genes in accordance with the present inventionin Dunaliella viridis can be accomplished by modifying the methods andvectors taught by Sun et al. as discussed herein. Briefly, Sun et al.,Gene, Vol. 377 (2006), pp. 140-149, reported the stable transformationof Dunaliella viridis with plasmid DNA. Using the transformation methodof electroporation, Sun introduced the plasmid pDVNR, encoding the fullDunaliella viridis nitrate reductase gene into mutant Dunaliella viridis(Dunaliella viridis NR-mutants.) The NR-mutants are incapable of growthwithout the use of nitrate as a source of nitrogen. Nitrate reductasecatalyzes the conversion of nitrate to nitrite. Prior to transformation,Dunaliella viridis NR-mutants were unable to propagate in culture mediumcomprising nitrate (NO₃ ⁻) as the sole nitrogen source. The expressionof the Dunaliella viridis NR gene product in NR-mutant Dunaliellaviridis was used as a selectable marker to rescue the nitrate metabolismdeficiency. Upon transformation with the pDVNR plasmid, NR-mutantDunaliella viridis stably expressing the Dunaliella viridis NR geneproduct were obtained that were able to grow on agar plates comprisingnitrate as the sole carbon source. Evaluation of the DNA of the stabletransformants was performed by Southern analysis. Selection andmaintenance of the transformed Dunaliella viridis (NR mutant) wasperformed on agar plates comprising 5 mM KNO₃. Sun also reported thepropagation of Dunaliella viridis and Dunaliella viridis NR mutants inliquid culture medium. Additional media suitable for propagation ofDunaliella viridis are reported by Gordillo et al., Journal of AppliedPhycology, Vol. 10:2 (1998), pp. 135-144 and by Moulton and Burford,Hydrobiologia, Vols. 204-205:1 (1990), pp. 401-408. Sun reported thatthe plasmid pDVNR and the promoter and 3′ UTR/terminator of theDunaliella viridis nitrate reductase gene were suitable to enableheterologous expression in Dunaliella viridis NR-mutants. Sun alsoreported that expression of the Dunaliella viridis nitrate reductasegene product was suitable for use as a selectable marker in Dunaliellaviridis NR-mutants.

In an embodiment of the present invention, vector pDVNR, comprising thenucleotide sequence encoding the Dunaliella viridis nitrate reductase(DvNR) gene product for use as a selectable marker, is constructed andmodified to further comprise a lipid biosynthesis pathway expressioncassette sequence, thereby creating a transformation vector. The lipidbiosynthesis pathway expression cassette encodes one or more lipidbiosynthesis pathway proteins selected Table 20, each protein-codingsequence codon-optimized for expression in Dunaliella viridis to reflectthe codon bias inherent in nuclear genes of Dunaliella viridis inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the DvNR promoter upstream of the protein-codingsequence and operably linked to the DvNR 3′UTR/terminator at the 3′region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Dunaliella viridis genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Dunaliella viridis NR mutants with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe DvNR gene product can be used as a selectable marker to rescue thenitrogen assimilation deficiency of Dunaliella viridis NR mutant strainsand to select for Dunaliella viridis NR-mutants stably expressing thetransformation vector. Growth media suitable for Dunaliella viridislipid production include, but are not limited to those discussed by Sunet al., Moulton and Burford, and Gordillo et al. Evaluation of fattyacid profiles of Dunaliella viridis lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 19 Engineering Dunaliella salina

Expression of recombinant genes in accordance with the present inventionin Dunaliella salina can be accomplished by modifying the methods andvectors taught by Geng et al. as discussed herein. Briefly, Geng et al.,Journal of Applied Phycology, Vol. (2003), pp. 451-456, reported thestable transformation of Dunaliella salina with plasmid DNA. Using thetransformation method of electroporation, Geng introduced thepUΩHBsAg-CAT plasmid into Dunaliella salina. pUΩHBsAg-CAT comprises ahepatitis B surface antigen (HBsAG) expression cassette comprisingsequence encoding the hepatitis B surface antigen operably linked to aZea mays ubi1 promoter upstream of the HBsAG protein-coding region andoperably linked to the 3′UTR/terminator of the Agrobacterium tumefaciensnopaline synthase gene (nos) downstream of the HBsAG protein-codingregion. pUΩHBsAg-CAT further comprised a chloramphenicol resistancecassette, comprising sequence encoding the chloramphenicolacetyltransferase (CAT) gene product, conferring resistance to theantibiotic chloramphenicol, operably linked to the simian virus 40promoter and enhancer. Prior to transformation with pUΩHBsAg-CAT,Dunaliella salina was unable to propagate on medium comprising 60 mg/Lchloramphenicol. Upon transformation with the pUΩHBsAg-CAT plasmid,transformants of Dunaliella salina were obtained that were propagated inselective culture medium comprising 60 mg/L chloramphenicol. Theexpression of the CAT gene product in Dunaliella salina enabledpropagation in the presence of 60 mg/L chloramphenicol, therebyestablishing the utility of the chloramphenicol resistance cassette asselectable marker for use in Dunaliella salina. Detectable activity ofthe HBsAg gene product indicated that ubi1 promoter and nos3′UTR/terminator are suitable for enabling gene expression in Dunaliellasalina. Evaluation of the genomic DNA of the stable transformants wasperformed by Southern analysis. Geng reported that selection andmaintenance of the transformed Dunaliella salina was performed on agarplates comprising Johnson's medium (J1, described by Borowitzka andBorowitzka (eds), Micro-algal Biotechnology. Cambridge University Press,Cambridge, pp. 460-461) with 60 mg/L chloramphenicol. Liquid propagationof Dunaliella salina was performed by Geng in J1 medium with 60 mg/Lchloramphenicol. Propagation of Dunaliella salina in media other than J1medium has been discussed (see Feng et al., Mol. Bio. Reports, Vol. 36(2009), pp. 1433-1439 and Borowitzka et al., Hydrobiologia, Vols.116-117:1 (1984), pp. 115-121). Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Dunaliella salina have been reported byFeng et al. Geng reported that the plasmid pUΩHBsAg-CAT, the ubi1promoter, and the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator are suitable to enable exogenous gene expression inDunaliella salina. In addition, Geng reported that the CAT resistancecassette encoded on pUΩHBsAg-CAT was suitable for use as a selectablemarker in Dunaliella salina.

In an embodiment of the present invention, vector pUΩHBsAg-CAT,comprising the nucleotide sequence encoding the CAT gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Dunaliella salina to reflect the codon bias inherentin nuclear genes of Dunaliella salina in accordance with Tables 19A-D.For each lipid biosynthesis pathway protein of Table 20, thecodon-optimized gene sequence can individually be operably linked to theubi1 promoter upstream of the protein-coding sequence and operablylinked to the Agrobacterium tumefaciens nopaline synthase gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Dunaliella salina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Dunaliella salinawith the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the CAT gene product can be used as a selectablemarker to select for Dunaliella salina transformed with thetransformation vector in, but not limited to, J1 medium comprisingchloramphenicol. Growth medium suitable for Dunaliella salina lipidproduction include, but are not limited to J1 medium and those culturemedia described by Feng et al. and Borowitzka et al. Evaluation of fattyacid profiles of Dunaliella salina lipids can be assessed throughstandard lipid extraction and analytical methods described herein.

Example 20 Engineering Gonium pectoral

Expression of recombinant genes in accordance with the present inventionin Gonium pectoral can be accomplished by modifying the methods andvectors taught by Lerche and Hallman et al. as discussed herein.Briefly, Lerche and Hallman et al., BMC Biotechnology, Volume 9:64,2009, reported the stable nuclear transformation of Gonium pectoralewith plasmid DNA. Using the transformation method of microprojectilebombardment, Lerche introduced the plasmid pPmr3 into Gonium pectorale.Plasmid pPmr3 comprised a paromomycin resistance cassette, comprising asequence encoding the aminoglycoside 3′-phosphotransferase (aphVIII)gene product (GenBank Accession No. AAB03856) of Streptomyces rimosusfor resistance to the antibiotic paromomycin, operably linked to theVolvox carteri hsp70A-rbcS3 hybrid promoter upstream of the aphVIIIprotein-coding region and operably linked to the 3′ UTR/terminator ofthe Volvox carteri rbcS3 gene downstream of the aphVIII protein-codingregion. Prior to transformation with pPmr3, Gonium pectorale was unableto propagate on medium comprising 0.06 ug/ml paromomycin. Upontransformation with pPmr3, transformants of Gonium pectorale wereobtained that were propagated in selective culture medium comprising0.75 and greater ug/ml paromomycin. The expression of the aphVIII geneproduct in Gonium pectorale enabled propagation in the presence of 0.75and greater ug/ml paromomycin, thereby establishing the utility of theparomomycin antibiotic resistance cassette as selectable marker for usein Gonium pectorale. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Lerche and Hallmanreported that selection and maintenance of the transformed Goniumpectorale was performed in liquid Jaworski's medium (20 mg/LCa(NO₃)₂.4H₂O, 12.4 mg/L KH₂PO₄, 50 mg/L MgSO₄.7H₂O, 15.9 mg/L NaHCO₃,2.25 mg/L EDTA-FeNa, 2.25 mg/L EDTA Na₂, 2.48 g/L H₃BO₃, 1.39 g/LMnCl₂.4H₂O, 1 mg/L (NH₄)₆MO₇O₂4.4H₂O, 0.04 mg/L vitamin B12, 0.04 mg/LThiamine-HCl, 0.04 mg/L biotin, 80 mg/L NaNO₃, 36 mg/L Na₄HPO₄.12H₂O)with 1.0 ug/ml paromomycin. Additional plasmids, promoters,3′UTR/terminators, and selectable markers suitable for enablingheterologous gene expression in Gonium pectorale are further discussedby Lerche and Hallman. Lerche and Hallman reported that the plasmidpPmr3, Volvox carteri hsp70A-rbcS3 hybrid promoter, and the 3′UTR/terminator of the Volvox carteri rbcS3 gene are suitable to enableexogenous gene expression in Gonium pectorale. In addition, Lerche andHallman reported that the paromomycin resistance cassette encoded pPmr3was suitable for use as a selectable marker in Gonium pectorale.

In an embodiment of the present invention, vector pPmr3, comprising thenucleotide sequence encoding the aphVIII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Gonium pectorale to reflect the codon bias inherent innuclear genes of Gonium pectorale in accordance with Tables 19A-D. Foreach lipid biosynthesis pathway protein of Table 20, the codon-optimizedgene sequence can individually be operably linked to the Volvox carterihsp70A-rbcS3 hybrid promoter upstream of the protein-coding sequence andoperably linked to the Volvox carteri rbcS3 gene 3′UTR/terminator at the3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Gonium pectorale genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Gonium pectorale with the transformation vectorcan be achieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of theaphVIII gene product can be used as a selectable marker to select forGonium pectorale transformed with the transformation vector in, but notlimited to, Jaworski's medium comprising paromomycin. Growth mediasuitable for Gonium pectorale lipid production include Jawaorski'smedium and media reported by Stein, American Journal of Botany, Vol.45:9 (1958), pp. 664-672. Evaluation of fatty acid profiles of Goniumpectorale lipids can be assessed through standard lipid extraction andanalytical methods described herein.

Example 21 Engineering Phaeodactylum tricornutum

Expression of recombinant genes in accordance with the present inventionin Phaeodactylum tricornutum can be accomplished by modifying themethods and vectors taught by Apt et al. as discussed herein. Briefly,Apt et al., Molecular and General Genetics, Vol. 252 (1996), pp.572-579, reported the stable nuclear transformation of Phaeodactylumtricornutum with vector DNA. Using the transformation technique ofmicroprojectile bombardment, Apt introduced the plasmid pfcpA intoPhaeodactylum tricornutum. Plasmid pfcpA comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics phleomycin and zeocin, operably linked to the promoter ofthe Phaeodactylum tricornutum fucoxanthin chlorophyll a binding proteingene (fcpA) upstream of the ble protein-coding region and operablylinked to the 3′ UTR/terminator of the Phaeodactylum tricornutum fcpAgene at the 3′ region, or downstream of the ble protein-coding region.Prior to transformation with pfcpA, Phaeodactylum tricornutum was unableto propagate on medium comprising 50 ug/ml zeocin. Upon transformationwith pfcpA, transformants of Phaeodactylum tricornutum were obtainedthat were propagated in selective culture medium comprising 50 ug/mlzeocin. The expression of the ble gene product in Phaeodactylumtricornutum enabled propagation in the presence of 50 ug/ml zeocin,thereby establishing the utility of the bleomycin antibiotic resistancecassette as selectable marker for use in Phaeodactylum tricornutum.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Apt reported that selection and maintenance of thetransformed Phaeodactylum tricornutum was performed on agar platescomprising LDM medium (as reported by Stan and Zeikus, Journal ofPhycology, Vol. 29, Supplement, (1993)) with 50 mg/L zeocin. Aptreported liquid propagation of Phaeodactylum tricornutum transformantsin LDM medium with 50 mg/L zeocin. Propagation of Phaeodactylumtricornutum in medium other than LDM medium has been discussed (byZaslayskaia et al., Science, Vol. 292 (2001), pp. 2073-2075, and byRadokovits et al., Metabolic Engineering, Vol. 13 (2011), pp. 89-95).Additional plasmids, promoters, 3′UTR/terminators, and selectablemarkers suitable for enabling heterologous gene expression inPhaeodactylum tricornutum have been reported in the same report by Aptet al., by Zaslayskaia et al., and by Radokovits et al.). Apt reportedthat the plasmid pfcpA, and the Phaeodactylum tricornutum fcpA promoterand 3′ UTR/terminator are suitable to enable exogenous gene expressionin Phaeodactylum tricornutum. In addition, Apt reported that thebleomycin resistance cassette encoded on pfcpA was suitable for use as aselectable marker in Phaeodactylum tricornutum.

In an embodiment of the present invention, vector pfcpA, comprising thenucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Phaeodactylum tricornutum to reflect the codon biasinherent in nuclear genes of Phaeodactylum tricornutum in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the Phaeodactylum tricornutum fcpA gene promoter upstream ofthe protein-coding sequence and operably linked to the Phaeodactylumtricornutum fcpA gene 3′UTR/terminator at the 3′ region, or downstream,of the protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Phaeodactylum tricornutumgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Phaeodactylumtricornutum genome (referenced in the publication by Bowler et al.,Nature, Vol. 456 (2008), pp. 239-244). Stable transformation ofPhaeodactylum tricornutum with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product canbe used as a marker to select for Phaeodactylum tricornutum transformedwith the transformation vector in, but not limited to, LDM mediumcomprising paromomycin. Growth medium suitable for Phaeodactylumtricornutum lipid production include, but are not limited to f/2 mediumas reported by Radokovits et al. Evaluation of fatty acid profiles ofPhaeodactylum tricornutum lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 22 Engineering Chaetoceros sp.

Expression of recombinant genes in accordance with the present inventionin Chaetoceros sp. can be accomplished by modifying the methods andvectors taught by Yamaguchi et al. as discussed herein. Briefly,Yamaguchi et al., Phycological Research, Vol. 59:2 (2011), pp. 113-119,reported the stable nuclear transformation of Chaetoceros sp. withplasmid DNA. Using the transformation method of microprojectilebombardment, Yamaguchi introduced the plasmid pTpfcp/nat intoChaetoceros sp. pTpfcp/nat comprised a nourseothricin resistancecassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Chaetoceros sp. was unable to propagate on mediumcomprising 500 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Chaetoceros sp. were obtained that werepropagated in selective culture medium comprising 500 ug/mlnourseothricin. The expression of the nat gene product in Chaetocerossp. enabled propagation in the presence of 500 ug/ml nourseothricin,thereby establishing the utility of the nourseothricin antibioticresistance cassette as selectable marker for use in Chaetoceros sp.Evaluation of the genomic DNA of the stable transformants was performedby Southern analysis. Yamaguchi reported that selection and maintenanceof the transformed Chaetoceros sp. was performed on agar platescomprising f/2 medium (as reported by Guilard, R. R., Culture ofPhytoplankton for feeding marine invertebrates, In Culture of MarineInvertebrate Animals, Smith and Chanley (eds) 1975, Plenum Press, NewYork, pp. 26-60) with 500 ug/ml nourseothricin. Liquid propagation ofChaetoceros sp. transformants, as performed by Yamaguchi, was carriedout in f/2 medium with 500 mg/L nourseothricin. Propagation ofChaetoceros sp. in additional culture medium has been reported (forexample in Napolitano et al., Journal of the World Aquaculture Society,Vol. 21:2 (1990), pp. 122-130, and by Volkman et al., Journal ofExperimental Marine Biology and Ecology, Vol. 128:3 (1989), pp.219-240). Additional plasmids, promoters, 3′UTR/terminators, andselectable markers suitable for enabling heterologous gene expression inChaetoceros sp. have been reported in the same report by Yamaguchi etal. Yamaguchi reported that the plasmid pTpfcp/nat, and theThalassiosira pseudonana fcp promoter and 3′ UTR/terminator are suitableto enable exogenous gene expression in Chaetoceros sp. In addition,Yamaguchi reported that the nourseothricin resistance cassette encodedon pTpfcp/nat was suitable for use as a selectable marker in Chaetocerossp.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in the closely-related Chaetoceroscompressum to reflect the codon bias inherent in nuclear genes ofChaetoceros compressum in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Thalassiosirapseudonana fcp gene promoter upstream of the protein-coding sequence andoperably linked to the Thalassiosira pseudonana fcp gene3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Chaetoceros sp. genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Chaetoceros sp.with the transformation vector is achieved through well-knowntransformation including microprojectile bombardment or other knownmethods. Activity of the nat gene product can be used as a selectablemarker to select for Chaetoceros sp. transformed with the transformationvector in, but not limited to, f/2 agar medium comprisingnourseothricin. Growth medium suitable for Chaetoceros sp. lipidproduction include, but are not limited to, f/2 medium, and thoseculture media discussed by Napolitano et al. and Volkman et al.Evaluation of fatty acid profiles of Chaetoceros sp lipids can beassessed through standard lipid extraction and analytical methodsdescribed herein.

Example 23 Engineering Cylindrotheca fusiformis

Expression of recombinant genes in accordance with the present inventionin Cylindrotheca fusiformis can be accomplished by modifying the methodsand vectors taught by Poulsen and Kroger et al. as discussed herein.Briefly, Poulsen and Kroger et al., FEBS Journal, Vol. 272 (2005), pp.3413-3423, reported the transformation of Cylindrotheca fusiformis withplasmid DNA. Using the transformation method of microprojectilebombardment, Poulsen and Kroger introduced the pCF-ble plasmid intoCylindrotheca fusiformis. Plasmid pCF-ble comprised a bleomycinresistance cassette, comprising sequence encoding the Streptoalloteichushindustanus Bleomycin binding protein (ble), for resistance to theantibiotics zeocin and phleomycin, operably linked to the Cylindrothecafusiformis fucozanthin chlorophyll a/c binding protein gene (fcpA,GenBank Accession No. AY125580) promoter upstream of the bleprotein-coding region and operably linked to the Cylindrothecafusiformis fcpA gene 3′UTR/terminator at the 3′ region (down-stream ofthe ble protein-coding region). Prior to transformation with pCF-ble,Cylindrotheca fusiformis was unable to propagate on medium comprising 1mg/ml zeocin. Upon transformation with pCF-ble, transformants ofCylindrotheca fusiformis were obtained that were propagated in selectiveculture medium comprising 1 mg/ml zeocin. The expression of the ble geneproduct in Cylindrotheca fusiformis enabled propagation in the presenceof 1 mg/ml zeocin, thereby establishing the utility of the bleomycinantibiotic resistance cassette as selectable marker for use inCylindrotheca fusiformis. Poulsen and Kroger reported that selection andmaintenance of the transformed Cylindrotheca fusiformis was performed onagar plates comprising artificial seawater medium with 1 mg/ml zeocin.Poulsen and Kroger reported liquid propagation of Cylindrothecafusiformis transformants in artificial seawater medium with 1 mg/mlzeocin. Propagation of Cylindrotheca fusiformis in additional culturemedium has been discussed (for example in Liang et al., Journal ofApplied Phycology, Vol. 17:1 (2005), pp. 61-65, and by Orcutt andPatterson, Lipids, Vol. 9:12 (1974), pp. 1000-1003). Additionalplasmids, promoters, and 3′UTR/terminators for enabling heterologousgene expression in Chaetoceros sp. have been reported in the same reportby Poulsen and Kroger. Poulsen and Kroger reported that the plasmidpCF-ble and the Cylindrotheca fusiformis fcp promoter and 3′UTR/terminator are suitable to enable exogenous gene expression inCylindrotheca fusiformis. In addition, Poulsen and Kroger reported thatthe bleomycin resistance cassette encoded on pCF-ble was suitable foruse as a selectable marker in Cylindrotheca fusiformis.

In an embodiment of the present invention, vector pCF-ble, comprisingthe nucleotide sequence encoding the ble gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Cylindrotheca fusiformis to reflect the codon biasinherent in nuclear genes of Cylindrotheca fusiformis in accordance withTables 19A-D. For each lipid biosynthesis pathway protein of Table 20,the codon-optimized gene sequence can individually be operably linked tothe Cylindrotheca fusiformis fcp gene promoter upstream of theprotein-coding sequence and operably linked to the Cylindrothecafusiformis fcp gene 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Cylindrotheca fusiformisgenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. Stable transformation ofCylindrotheca fusiformis with the transformation vector is achievedthrough well-known transformation techniques including microprojectilebombardment or other known methods. Activity of the ble gene product canbe used as a selectable marker to select for Cylindrotheca fusiformistransformed with the transformation vector in, but not limited to,artificial seawater agar medium comprising zeocin. Growth media suitablefor Cylindrotheca fusiformis lipid production include, but are notlimited to, artificial seawater and those media reported by Liang et al.and Orcutt and Patterson. Evaluation of fatty acid profiles ofCylindrotheca fusiformis lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 24 Engineering Amphidinium sp.

Expression of recombinant genes in accordance with the present inventionin Amphidinium sp. can be accomplished by modifying the methods andvectors taught by ten Lohuis and Miller et al. as discussed herein.Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol. 13:3(1998), pp. 427-435, reported the stable transformation of Amphidiniumsp. with plasmid DNA. Using the transformation technique of agitation inthe presence of silicon carbide whiskers, ten Lohuis introduced theplasmid pMT NPT/GUS into Amphidinium sp. pMT NPT/GUS comprised aneomycin resistance cassette, comprising sequence encoding the neomycinphosphotransferase II (nptII) gene product (GenBank Accession No.AAL92039) operably linked to the Agrobacterium tumefaciens nopalinesynthase (nos) gene promoter upstream, or 5′ of the nptII protein-codingregion and operably linked to the 3′ UTR/terminator of the nos gene atthe 3′ region (down-stream of the nptII protein-coding region). ThenptII gene product confers resistance to the antibiotic G418. The pMTNPT/GUS plasmid further comprised sequence encoding a beta-glucuronidase(GUS) reporter gene product operably-linked to a CaMV 35S promoter andfurther operably linked to the CaMV 35S 3′ UTR/terminator. Prior totransformation with pMT NPT/GUS, Amphidinium sp. was unable to bepropagated on medium comprising 3 mg/ml G418. Upon transformation withpMT NPT/GUS, transformants of Amphidinium sp. were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Amphidinium sp. enabledpropagation in the presence of 3 mg/ml G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Amphidinium sp. Detectable activity of the GUSreporter gene indicated that CaMV 35S promoter and 3′UTR are suitablefor enabling gene expression in Amphidinium sp. Evaluation of thegenomic DNA of the stable transformants was performed by Southernanalysis. ten Lohuis and Miller reported liquid propagation ofAmphidinium sp transformants in medium comprising seawater supplementedwith F/2 enrichment solution (provided by the supplier Sigma) and 3mg/ml G418 as well as selection and maintenance of Amphidinium sp.transformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Amphidinium sp. inadditional culture medium has been reported (for example in Mansour etal., Journal of Applied Phycology, Vol. 17:4 (2005) pp. 287-v300). Anadditional plasmid, comprising additional promoters, 3′UTR/terminators,and a selectable marker for enabling heterologous gene expression inAmphidinium sp. have been reported in the same report by ten Lohuis andMiller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUS andthe promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Amphidinium sp. Inaddition, ten Lohuis and Miller reported that the neomycin resistancecassette encoded on pMT NPT/GUS was suitable for use as a selectablemarker in Amphidinium sp.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Amphidinium sp. to reflect the codonbias inherent in nuclear genes of the closely-related species,Amphidinium carterae in accordance with Tables 19A-D. For each lipidbiosynthesis pathway protein of Table 20, the codon-optimized genesequence can individually be operably linked to the Agrobacteriumtumefaciens nopaline synthase (nos) gene promoter upstream of theprotein-coding sequence and operably linked to the nos 3′UTR/terminatorat the 3′ region, or downstream, of the protein-coding sequence. Thetransformation construct may additionally comprise homology regions tothe Amphidinium sp. genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Amphidinium sp. with the transformation vectoris achieved through well-known transformation techniques includingsilicon fibre-mediated microinjection or other known methods. Activityof the nptII gene product can be used as a selectable marker to selectfor Amphidinium sp. transformed with the transformation vector in, butnot limited to, seawater agar medium comprising G418. Growth mediasuitable for Amphidinium sp. lipid production include, but are notlimited to, artificial seawater and those media reported by Mansour etal. and ten Lohuis and Miller. Evaluation of fatty acid profiles ofAmphidinium sp. lipids can be assessed through standard lipid extractionand analytical methods described herein.

Example 25 Engineering Symbiodinium microadriacticum

Expression of recombinant genes in accordance with the present inventionin Symbiodinium microadriacticum can be accomplished by modifying themethods and vectors taught by ten Lohuis and Miller et al. as discussedherein. Briefly, ten Lohuis and Miller et al., The Plant Journal, Vol.13:3 (1998), pp. 427-435, reported the stable transformation ofSymbiodinium microadriacticum with plasmid DNA. Using the transformationtechnique of silicon fibre-mediated microinjection, ten Lohuisintroduced the plasmid pMT NPT/GUS into Symbiodinium microadriacticum.pMT NPT/GUS comprised a neomycin resistance cassette, comprisingsequence encoding the neomycin phosphotransferase II (nptII) geneproduct (GenBank Accession No. AAL92039) operably linked to theAgrobacterium tumefaciens nopaline synthase (nos) gene promoterupstream, or 5′ of the nptII protein-coding region and operably linkedto the 3′ UTR/terminator of the nos gene at the 3′ region (down-streamof the nptII protein-coding region). The nptII gene product confersresistance to the antibiotic G418. The pMT NPT/GUS plasmid furthercomprised sequence encoding a beta-glucuronidase (GUS) reporter geneproduct operably-linked to a CaMV 35S promoter and further operablylinked to the CaMV 35S 3′ UTR/terminator. Prior to transformation withpMT NPT/GUS, Symbiodinium microadriacticum was unable to be propagatedon medium comprising 3 mg/ml G418. Upon transformation with pMT NPT/GUS,transformants of Symbiodinium microadriacticum were obtained that werepropagated in selective culture medium comprising 3 mg/ml G418. Theexpression of the nptII gene product in Symbiodinium microadriacticumenabled propagation in the presence of 3 mg/ml G418, therebyestablishing the utility of the neomycin antibiotic resistance cassetteas selectable marker for use in Symbiodinium microadriacticum.Detectable activity of the GUS reporter gene indicated that CaMV 35Spromoter and 3′UTR are suitable for enabling gene expression inSymbiodinium microadriacticum. Evaluation of the genomic DNA of thestable transformants was performed by Southern analysis. ten Lohuis andMiller reported liquid propagation of Symbiodinium microadriacticumtransformants in medium comprising seawater supplemented with F/2enrichment solution (provided by the supplier Sigma) and 3 mg/ml G418 aswell as selection and maintenance of Symbiodinium microadriacticumtransformants on agar medium comprising seawater supplemented with F/2enrichment solution and 3 mg/ml G418. Propagation of Symbiodiniummicroadriacticum in additional culture medium has been discussed (forexample in Iglesias-Prieto et al., Proceedings of the National Academyof Sciences, Vol. 89:21 (1992) pp. 10302-10305). An additional plasmid,comprising additional promoters, 3′UTR/terminators, and a selectablemarker for enabling heterologous gene expression in Symbiodiniummicroadriacticum have been discussed in the same report by ten Lohuisand Miller. ten Lohuis and Miller reported that the plasmid pMT NPT/GUSand the promoter and 3′ UTR/terminator of the nos and CaMV 35S genes aresuitable to enable exogenous gene expression in Symbiodiniummicroadriacticum. In addition, ten Lohuis and Miller reported that theneomycin resistance cassette encoded on pMT NPT/GUS was suitable for useas a selectable marker in Symbiodinium microadriacticum.

In an embodiment of the present invention, vector pMT NPT/GUS,comprising the nucleotide sequence encoding the nptII gene product foruse as a selectable marker, is constructed and modified to furthercomprise a lipid biosynthesis pathway expression cassette sequence,thereby creating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected Table 20, each protein-coding sequence codon-optimizedfor expression in Symbiodinium microadriacticum to reflect the codonbias inherent in nuclear genes of Symbiodinium microadriacticum inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Agrobacterium tumefaciens nopaline synthase(nos) gene promoter upstream of the protein-coding sequence and operablylinked to the nos 3′UTR/terminator at the 3′ region, or downstream, ofthe protein-coding sequence. The transformation construct mayadditionally comprise homology regions to the Symbiodiniummicroadriacticum genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Symbiodinium microadriacticum with thetransformation vector is achieved through well-known transformationtechniques including silicon fibre-mediated microinjection or otherknown methods. Activity of the nptII gene product can be used as aselectable marker to select for Symbiodinium microadriacticumtransformed with the transformation vector in, but not limited to,seawater agar medium comprising G418. Growth media suitable forSymbiodinium microadriacticum lipid production include, but are notlimited to, artificial seawater and those media reported byIglesias-Prieto et al. and ten Lohuis and Miller. Evaluation of fattyacid profiles of Symbiodinium microadriacticum lipids can be assessedthrough standard lipid extraction and analytical methods describedherein.

Example 26 Engineering Nannochloropsis sp.

Expression of recombinant genes in accordance with the present inventionin Nannochloropsis sp. W2J3B can be accomplished by modifying themethods and vectors taught by Kilian et al. as discussed herein.Briefly, Kilian et al., Proceedings of the National Academy of Sciences,Vol. 108:52 (2011) pp. 21265-21269, reported the stable nucleartransformation of Nannochloropsis with a transformation construct. Usingthe transformation method of electroporation, Kilian introduced thetransformation construct C2 into Nannochloropsis sp. W2J3B. The C2transformation construct comprised a bleomycin resistance cassette,comprising the coding sequence for the Streptoalloteichus hindustanusBleomycin binding protein (ble), for resistance to the antibioticsphleomycin and zeocin, operably linked to and the promoter of theNannochloropsis sp. W2J3B violaxanthin/chlorophyll a-binding proteingene VCP2 upstream of the ble protein-coding region and operably linkedto the 3′UTR/terminator of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding gene VCP1 downstream of the bleprotein-coding region. Prior to transformation with C2, Nannochloropsissp. W2J3B was unable to propagate on medium comprising 2 ug/ml zeocin.Upon transformation with C2, transformants of Nannochloropsis sp. W2J3Bwere obtained that were propagated in selective culture mediumcomprising 2 ug/ml zeocin. The expression of the ble gene product inNannochloropsis sp. W2J3B enabled propagation in the presence of 2 ug/mlzeocin, thereby establishing the utility of the bleomycin antibioticresistance cassette as selectable marker for use in Nannochloropsis.Evaluation of the genomic DNA of the stable transformants was performedby PCR. Kilian reported liquid propagation of Nannochloropsis sp. W2J3Btransformants in F/2 medium (reported by Guilard and Ryther, CanadianJournal of Microbiology, Vol. 8 (1962), pp. 229-239) comprising fivefoldlevels of trace metals, vitamins, and phosphate solution, and furthercomprising 2 ug/ml zeocin. Kilian also reported selection andmaintenance of Nannochloropsis sp. W2J3B transformants on agar F/2medium comprising artificial seawater 2 mg/ml zeocin. Propagation ofNannochloropsis in additional culture medium has been discussed (forexample in Chiu et al., Bioresour Technol., Vol. 100:2 (2009), pp.833-838 and Pal et al., Applied Microbiology and Biotechnology, Vol.90:4 (2011), pp. 1429-1441). Additional transformation constructs,comprising additional promoters and 3′UTR/terminators for enablingheterologous gene expression in Nannochloropsis sp. W2J3B and selectablemarkers for selection of transformants have been described in the samereport by Kilian. Kilian reported that the transformation construct C2and the promoter of the Nannochloropsis sp. W2J3Bviolaxanthin/chlorophyll a-binding protein gene VCP2 and 3′UTR/terminator of the Nannochloropsis sp. W2J3B violaxanthin/chlorophylla-binding protein gene VCP1 are suitable to enable exogenous geneexpression in Nannochloropsis sp. W2J3B. In addition, Kilian reportedthat the bleomycin resistance cassette encoded on C2 was suitable foruse as a selectable marker in Nannochloropsis sp. W2J3B.

In an embodiment of the present invention, transformation construct C2,comprising the nucleotide sequence encoding the ble gene product for useas a selectable marker, is constructed and modified to further comprisea lipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Nannochloropsis sp. W2J3B to reflectthe codon bias inherent in nuclear genes of Nannochloropsis sp. inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Nannochloropsis sp. W2J3B VCP2 gene promoterupstream of the protein-coding sequence and operably linked to theNannochloropsis sp. W2J3B VCP1 gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to theNannochloropsis sp. W2J3B genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic sites of endogenous lipid biosynthesis pathway genes.Stable transformation of Nannochloropsis sp. W2J3B with thetransformation vector is achieved through well-known transformationtechniques including electroporation or other known methods. Activity ofthe ble gene product can be used as a selectable marker to select forNannochloropsis sp. W2J3B transformed with the transformation vector in,but not limited to, F/2 medium comprising zeocin. Growth media suitablefor Nannochloropsis sp. W2J3B lipid production include, but are notlimited to, F/2 medium and those media reported by Chiu et al. and Palet al. Evaluation of fatty acid profiles of Nannochloropsis sp. W2J3Blipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 27 Engineering Cyclotella cryptica

Expression of recombinant genes in accordance with the present inventionin Cyclotella cryptica can be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Cyclotella cryptica with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Cyclotella cryptica. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Cyclotella cryptica was unable to propagate on 50%artificial seawater medium comprising 100 ug/ml G418. Upontransformation with pACCNPT5.1, transformants of Cyclotella crypticawere obtained that were propagated in selective 50% artificial seawatermedium comprising 100 ug/ml G418. The expression of the nptII geneproduct in Cyclotella cryptica enabled propagation in the presence of100 ug/ml G418, thereby establishing the utility of the neomycinantibiotic resistance cassette as selectable marker for use inCyclotella cryptica. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Dunahay reportedliquid propagation of Cyclotella cryptica in artificial seawater medium(ASW, as discussed by Brown, L., Phycologia, Vol. 21 (1982), pp.408-410) supplemented with 1.07 mM sodium silicate and with 100 ug/mlG418. Dunahay also reported selection and maintenance of Cyclotellacryptica transformants on agar plates comprising ASW medium with 100ug/ml G418. Propagation of Cyclotella cryptica in additional culturemedium has been discussed (for example in Sriharan et al., AppliedBiochemistry and Biotechnology, Vol. 28-29:1 (1991), pp. 317-326 andPahl et al., Journal of Bioscience and Bioengineering, Vol. 109:3(2010), pp. 235-239). Dunahay reported that the plasmid pACCNPT5.1 andthe promoter of the Cyclotella cryptica acetyl-CoA carboxylase (ACCase)gene are suitable to enable exogenous gene expression in Cyclotellacryptica. In addition, Dunahay reported that the neomycin resistancecassette encoded on pACCNPT5.1 was suitable for use as a selectablemarker in Cyclotella cryptica.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Cyclotella cryptica to reflect thecodon bias inherent in nuclear genes of Cyclotella cryptica inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Cyclotella cryptica ACCase promoter upstreamof the protein-coding sequence and operably linked to the Cyclotellacryptica ACCase 3′UTR/terminator at the 3′ region, or downstream, of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Cyclotella cryptica genome for targetedgenomic integration of the transformation vector. Homology regions maybe selected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. Stable transformation of Cyclotella crypticawith the transformation vector is achieved through well-knowntransformation techniques including microprojectile bombardment or otherknown methods. Activity of the nptII gene product can be used as amarker to select for for Cyclotella cryptica transformed with thetransformation vector in, but not limited to, agar ASW medium comprisingG418. Growth media suitable for Cyclotella cryptica lipid productioninclude, but are not limited to, ASW medium and those media reported bySriharan et al., 1991 and Pahl et al. Evaluation of fatty acid profilesof Cyclotella cryptica lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 28 Engineering Navicula saprophila

Expression of recombinant genes in accordance with the present inventionin Navicula saprophila can be accomplished by modifying the methods andvectors taught by Dunahay et al. as discussed herein. Briefly, Dunahayet al., Journal of Phycology, Vol. 31 (1995), pp. 1004-1012, reportedthe stable transformation of Navicula saprophila with plasmid DNA. Usingthe transformation method of microprojectile bombardment, Dunahayintroduced the plasmid pACCNPT5.1 into Navicula saprophila. PlasmidpACCNPT5.1 comprised a neomycin resistance cassette, comprising thecoding sequence of the neomycin phosphotransferase II (nptII) geneproduct operably linked to the promoter of the Cyclotella crypticaacetyl-CoA carboxylase (ACCase) gene (GenBank Accession No. L20784)upstream of the nptII coding-region and operably linked to the3′UTR/terminator of the Cyclotella cryptica ACCase gene at the 3′ region(downstream of the nptII coding-region). The nptII gene product confersresistance to the antibiotic G418. Prior to transformation withpACCNPT5.1, Navicula saprophila was unable to propagate on artificialseawater medium comprising 100 ug/ml G418. Upon transformation withpACCNPT5.1, transformants of Navicula saprophila were obtained that werepropagated in selective artificial seawater medium comprising 100 ug/mlG418. The expression of the nptII gene product in Navicula saprophilaenabled propagation in the presence of G418, thereby establishing theutility of the neomycin antibiotic resistance cassette as selectablemarker for use in Navicula saprophila. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Dunahayreported liquid propagation of Navicula saprophila in artificialseawater medium (ASW, as discussed by Brown, L., Phycologia, Vol. 21(1982), pp. 408-410) supplemented with 1.07 mM sodium silicate and with100 ug/ml G418. Dunahay also reported selection and maintenance ofNavicula saprophila transformants on agar plates comprising ASW mediumwith 100 ug/ml G418. Propagation of Navicula saprophila in additionalculture medium has been discussed (for example in Tadros and Johansen,Journal of Phycology, Vol. 24:4 (1988), pp. 445-452 and Sriharan et al.,Applied Biochemistry and Biotechnology, Vol. 20-21:1 (1989), pp.281-291). Dunahay reported that the plasmid pACCNPT5.1 and the promoterof the Cyclotella cryptica acetyl-CoA carboxylase (ACCase) gene aresuitable to enable exogenous gene expression in Navicula saprophila. Inaddition, Dunahay reported that the neomycin resistance cassette encodedon pACCNPT5.1 was suitable for use as a selectable marker in Naviculasaprophila.

In an embodiment of the present invention, vector pACCNPT5.1, comprisingthe nucleotide sequence encoding the nptII gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Navicula saprophila to reflect thecodon bias inherent in nuclear genes of the closely-related Naviculapelliculosa in accordance with Tables 19A-D. For each lipid biosynthesispathway protein of Table 20, the codon-optimized gene sequence canindividually be operably linked to the Cyclotella cryptica ACCase genepromoter upstream of the protein-coding sequence and operably linked tothe Cyclotella cryptica ACCase gene 3′UTR/terminator at the 3′ region,or downstream, of the protein-coding sequence. The transformationconstruct may additionally comprise homology regions to the Naviculasaprophila genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. Stabletransformation of Navicula saprophila with the transformation vector isachieved through well-known transformation techniques includingmicroprojectile bombardment or other known methods. Activity of thenptII gene product can be used as a selectable marker to select forNavicula saprophila transformed with the transformation vector in, butnot limited to, agar ASW medium comprising G418. Growth media suitablefor Navicula saprophila lipid production include, but are not limitedto, ASW medium and those media reported by Sriharan et al. 1989 andTadros and Johansen. Evaluation of fatty acid profiles of Naviculasaprophila lipids can be assessed through standard lipid extraction andanalytical methods described herein.

Example 29 Engineering Thalassiosira pseudonana

Expression of recombinant genes in accordance with the present inventionin Thalassiosira pseudonana can be accomplished by modifying the methodsand vectors taught by Poulsen et al. as discussed herein. Briefly,Poulsen et al., Journal of Phycology, Vol. 42 (2006), pp. 1059-1065,reported the stable transformation of Thalassiosira pseudonana withplasmid DNA. Using the transformation method of microprojectilebombardment, Poulsen introduced the plasmid pTpfcp/nat in toThalassiosira pseudonana. pTpfcp/nat comprised a nourseothricinresistance cassette, comprising sequence encoding the nourseothricinacetyltransferase (nat) gene product (GenBank Accession No. AAC60439)operably linked to the Thalassiosira pseudonana fucoxanthin chlorophylla/c binding protein gene (fcp) promoter upstream of the natprotein-coding region and operably linked to the Thalassiosirapseudonana fcp gene 3′ UTR/terminator at the 3′ region (downstream ofthe nat protein coding-sequence). The nat gene product confersresistance to the antibiotic nourseothricin. Prior to transformationwith pTpfcp/nat, Thalassiosira pseudonana was unable to propagate onmedium comprising 10 ug/ml nourseothricin. Upon transformation withpTpfcp/nat, transformants of Thalassiosira pseudonana were obtained thatwere propagated in selective culture medium comprising 100 ug/mlnourseothricin. The expression of the nat gene product in Thalassiosirapseudonana enabled propagation in the presence of 100 ug/mlnourseothricin, thereby establishing the utility of the nourseothricinantibiotic resistance cassette as selectable marker for use inThalassiosira pseudonana. Evaluation of the genomic DNA of the stabletransformants was performed by Southern analysis. Poulsen reported thatselection and maintenance of the transformed Thalassiosira pseudonanawas performed in liquid culture comprising modified ESAW medium (asdiscussed by Harrison et al., Journal of Phycology, Vol. 16 (1980), pp.28-35) with 100 ug/ml nourseothricin. Propagation of Thalassiosirapseudonana in additional culture medium has been discussed (for examplein Volkman et al., Journal of Experimental Marine Biology and Ecology,Vol. 128:3 (1989), pp. 219-240). An additional plasmid, comprisingadditional selectable markers suitable for use in Thalassiosirapseudonana has been discussed in the same report by Poulsen. Poulsenreported that the plasmid pTpfcp/nat, and the Thalassiosira pseudonanafcp promoter and 3′ UTR/terminator are suitable to enable exogenous geneexpression in Thalassiosira pseudonana. In addition, Poulsen reportedthat the nourseothricin resistance cassette encoded on pTpfcp/nat wassuitable for use as a selectable marker in Thalassiosira pseudonana.

In an embodiment of the present invention, vector pTpfcp/nat, comprisingthe nucleotide sequence encoding the nat gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Thalassiosira pseudonana to reflectthe codon bias inherent in nuclear genes of Thalassiosira pseudonana inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Thalassiosira pseudonana fcp gene promoterupstream of the protein-coding sequence and operably linked to theThalassiosira pseudonana fcp gene 3′ UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Thalassiosirapseudonana genome for targeted genomic integration of the transformationvector. Homology regions may be selected to disrupt one or more genomicsites of endogenous lipid biosynthesis pathway genes. One skilled in theart can identify such homology regions within the sequence of theThalassiosira pseudonana genome (referenced in the publication byArmbrust et al., Science, Vol. 306: 5693 (2004): pp. 79-86). Stabletransformation of Thalassiosira pseudonana with the transformationvector is achieved through well-known transformation techniquesincluding microprojectile bombardment or other known methods. Activityof the nat gene product can be used as a marker to select forThalassiosira pseudonana transformed with the transformation vector inbut not limited to, ESAW agar medium comprising nourseothricin. Growthmedia suitable for Thalassiosira pseudonana lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Volkman et al. and Harrison et al. Evaluation of fatty acid profilesof Thalassiosira pseudonana lipids can be assessed through standardlipid extraction and analytical methods described herein.

Example 30 Engineering Chlamydomonas reinhardtii

Expression of recombinant genes in accordance with the present inventionin Chlamydomonas reinhardtii can be accomplished by modifying themethods and vectors taught by Cerutti et al. as discussed herein.Briefly, Cerutti et al., Genetics, Vol. 145:1 (1997), pp. 97-110,reported the stable nuclear transformation of Chlamydomonas reinhardtiiwith a transformation vector. Using the transformation method ofmicroprojectile bombardment, Cerutti introduced transformation constructP[1030] into Chlamydomonas reinhardtii. Construct P[1030] comprised aspectinomycin resistance cassette, comprising sequence encoding theaminoglucoside 3″-adenyltransferase (aadA) gene product operably linkedto the Chlamydomonas reinhardtii ribulose-1,5-bisphosphatecarboxylase/oxygenase small subunit gene (RbcS2, GenBank Accession No.X04472) promoter upstream of the aadA protein-coding region and operablylinked to the Chlamydomonas reinhardtii RbcS2 gene 3′ UTR/terminator atthe 3′ region (downstream of the aadA protein coding-sequence). The aadAgene product confers resistance to the antibiotic spectinomycin. Priorto transformation with P[1030], Chlamydomonas reinhardtii was unable topropagate on medium comprising 90 ug/ml spectinomycin. Upontransformation with P[1030], transformants of Chlamydomonas reinhardtiiwere obtained that were propagated in selective culture mediumcomprising 90 ug/ml spectinomycin, thereby establishing the utility ofthe spectinomycin antibiotic resistance cassette as a selectable markerfor use in Chlamydomonas reinhardtii. Evaluation of the genomic DNA ofthe stable transformants was performed by Southern analysis. Ceruttireported that selection and maintenance of the transformed Chlamydomonasreinhardtii was performed on agar plates comprisingTris-acetate-phosphate medium (TAP, as described by Harris, TheChlamydomonas Sourcebook, Academic Press, San Diego, 1989) with 90 ug/mlspectinomycin. Cerutti additionally reported propagation ofChlamydomonas reinhardtii in TAP liquid culture with 90 ug/mlspectinomycin. Propagation of Chlamydomonas reinhardtii in alternativeculture medium has been discussed (for example in Dent et al., AfricanJournal of Microbiology Research, Vol. 5:3 (2011), pp. 260-270 andYantao et al., Biotechnology and Bioengineering, Vol. 107:2 (2010), pp.258-268). Additional constructs, comprising additional selectablemarkers suitable for use in Chlamydomonas reinhardtii as well asnumerous regulatory sequences, including protomers and 3′ UTRs suitablefor promoting heterologous gene expression in Chlamydomonas reinhardtiiare known in the art and have been discussed (for a review, seeRadakovits et al., Eurkaryotic Cell, Vol. 9:4 (2010), pp. 486-501).Cerutti reported that the transformation vector P[1030] and theChlamydomonas reinhardtii promoter and 3′ UTR/terminator are suitable toenable exogenous gene expression in Chlamydomonas reinhardtii. Inaddition, Cerutti reported that the spectinomycin resistance cassetteencoded on P[1030] was suitable for use as a selectable marker inChlamydomonas reinhardtii.

In an embodiment of the present invention, vector P[1030], comprisingthe nucleotide sequence encoding the aadA gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Chlamydomonas reinhardtii to reflectthe codon bias inherent in nuclear genes of Chlamydomonas reinhardtii inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Chlamydomonas reinhardtii RbcS2 promoterupstream of the protein-coding sequence and operably linked to theChlamydomonas reinhardtii RbcS2 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Chlamydomonasreinhardtii genome for targeted genomic integration of thetransformation vector. Homology regions may be selected to disrupt oneor more genomic site of an endogenous lipid biosynthesis pathway gene.One skilled in the art can identify such homology regions within thesequence of the Chlamydomonas reinhardtii genome (referenced in thepublication by Merchant et al., Science, Vol. 318:5848 (2007), pp.245-250). Stable transformation of Chlamydomonas reinhardtii with thetransformation vector is achieved through well-known transformationtechniques including microprojectile bombardment or other known methods.Activity of the aadA gene product can be used as a marker to select forChlamydomonas reinhardtii transformed with the transformation vector on,but not limited to, TAP agar medium comprising spectinomycin. Growthmedia suitable for Chlamydomonas reinhardtii lipid production include,but are not limited to, ESAW medium, and those culture media discussedby Yantao et al. and Dent et al. Evaluation of fatty acid profiles ofChlamydomonas reinhardtii lipids can be assessed through standard lipidextraction and analytical methods described herein.

Example 31 Engineering Yarrowia lipolytica

Expression of recombinant genes in accordance with the present inventionin Yarrowia lipolytica can be accomplished by modifying the methods andvectors taught by Fickers et al. as discussed herein. Briefly, Fickerset al., Journal of Microbiological Methods, Vol. 55 (2003), pp. 727-737,reported the stable nuclear transformation of Yarrowia lipolytica withplasmid DNA. Using a lithium acetate transformation method, Fickersintroduced the plasmid JMP123 into Yarrowia lipolytica. Plasmid JMP123comprised a hygromycin B resistance cassette, comprising sequenceencoding the hygromycin B phosphotransferase gene product (hph),operably-linked to the Yarrowia lipolytica LIP2 gene promoter (GenBankAccession No. AJ012632) upstream of the hph protein-coding region andoperably linked to the Yarrowia lipolytica LIP2 gene 3′UTR/terminatordownstream of the hph protein-coding region. Prior to transformationwith JMP123, Yarrowia lipolytica were unable to propagate on mediumcomprising 100 ug/ml hygromycin. Upon transformation with JMP123,transformed Yarrowia lipolytica were obtained that were able topropagate on medium comprising 100 ug/ml hygromycin, therebyestablishing the hygromycin B antibiotic resistance cassette as aselectable marker for use in Yarrowia lipolytica. The nucleotidesequence provided on JMP123 of the promoter and 3′UTR/terminator of theYarrowia lipolytica LIP2 gene served as donor sequences for homologousrecombination of the hph coding sequence into the LIP2 locus. Evaluationof the genomic DNA of the stable transformants was performed bySouthern. Fickers reported that selection and maintenance of thetransformed Yarrowia lipolytica was performed on agar plates comprisingstandard YPD medium (Yeast Extract Peptone Dextrose) with 100 ug/mlhygromycin. Liquid culturing of transformed Yarrowia lipolytica wasperformed in YPD medium with hygromycin. Other media and techniques usedfor culturing Yarrowia lipolytica have been reported and numerous otherplasmids, promoters, 3′ UTRs, and selectable markers for use in Yarrowialipolytica have been reported (for example see Pignede et al., Appliedand Environmental Biology, Vol. 66:8 (2000), pp. 3283-3289, Chuang etal., New Biotechnology, Vol. 27:4 (2010), pp. 277-282, and Barth andGaillardin, (1996), In: K, W. (Ed.), Nonconventional Yeasts inBiotechnology. Sprinter-Verlag, Berlin-Heidelber, pp. 313-388). Fickersreported that the transformation vector JMP123 and the Yarrowialipolytica LIP2 gene promoter and 3′ UTR/terminator are suitable toenable heterologous gene expression in Yarrowia lipolytica. In addition,Fickers reported that the hygromycin resistance cassette encoded onJMP123 was suitable for use as a selectable marker in Yarrowialipolytica.

In an embodiment of the present invention, vector JMP123, comprising thenucleotide sequence encoding the hph gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Yarrowia lipolytica to reflect thecodon bias inherent in nuclear genes of Yarrowia lipolytica inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the Yarrowia lipolytica LIP2 gene promoterupstream of the protein-coding sequence and operably linked to theYarrowia lipolytica LIP2 gene 3′UTR/terminator at the 3′ region, ordownstream, of the protein-coding sequence. The transformation constructmay additionally comprise homology regions to the Yarrowia lipolyticagenome for targeted genomic integration of the transformation vector.Homology regions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Yarrowialipolytica genome (referenced in the publication by Dujun et al.,Nature, Vol. 430 (2004), pp. 35-44). Stable transformation of Yarrowialipolytica with the transformation vector is achieved through well-knowntransformation techniques including lithium acetate transformation orother known methods. Activity of the hph gene product can be used as amarker to select for Yarrowia lipolytica transformed with thetransformation vector on, but not limited to, YPD medium comprisinghygromycin. Growth media suitable for Yarrowia lipolytica lipidproduction include, but are not limited to, YPD medium, and thoseculture media described by Chuang et al. Evaluation of fatty acidprofiles of Yarrowia lipolytica lipids can be assessed through standardlipid extraction and analytical methods described herein.

Example 32 Engineering Mortierella alpine

Expression of recombinant genes in accordance with the present inventionin Mortierella alpine can be accomplished by modifying the methods andvectors taught by Mackenzie et al. as discussed herein. Briefly,Mackenzie et al., Applied and Environmental Microbiology, Vol. 66(2000), pp. 4655-4661, reported the stable nuclear transformation ofMortierella alpina with plasmid DNA. Using a protoplast transformationmethod, MacKenzie introduced the plasmid pD4 into Mortierella alpina.Plasmid pD4 comprised a hygromycin B resistance cassette, comprisingsequence encoding the hygromycin B phosphotransferase gene product(hpt), operably-linked to the Mortierella alpina histone H4.1 genepromoter (GenBank Accession No. AJ249812) upstream of the hptprotein-coding region and operably linked to the Aspergillus nidulansN-(5′-phosphoribosyl)anthranilate isomerase (trpC) gene 3′UTR/terminatordownstream of the hpt protein-coding region. Prior to transformationwith pD4, Mortierella alpina were unable to propagate on mediumcomprising 300 ug/ml hygromycin. Upon transformation with pD4,transformed Mortierella alpina were obtained that were propagated onmedium comprising 300 ug/ml hygromycin, thereby establishing thehygromycin B antibiotic resistance cassette as a selectable marker foruse in Mortierella alpina. Evaluation of the genomic DNA of the stabletransformants was performed by Southern. Mackenzie reported thatselection and maintenance of the transformed Mortierella alpina wasperformed on PDA (potato dextrose agar) medium comprising hygromycin.Liquid culturing of transformed Mortierella alpina by Mackenzie wasperformed in PDA medium or in S2GYE medium (comprising 5% glucose, 0.5%yeast extract, 0.18% NH₄SO₄, 0.02% MgSO₄-7H₂O, 0.0001% FeCl₃-6H₂O, 0.1%,trace elements, 10 mM K₂HPO₄—NaH₂PO₄), with hygromycin. Other media andtechniques used for culturing Mortierella alpina have been reported andother plasmids, promoters, 3′ UTRs, and selectable markers for use inMortierella alpina have been reported (for example see Ando et al.,Applied and Environmental Biology, Vol. 75:17 (2009) pp. 5529-35 and Luet al., Applied Biochemistry and Biotechnology, Vol. 164:7 (2001), pp.979-90). Mackenzie reported that the transformation vector pD4 and theMortierella alpina histone H4.1 promoter and A. nidulans trpC gene 3′UTR/terminator are suitable to enable heterologous gene expression inMortierella alpina. In addition, Mackenzie reported that the hygromycinresistance cassette encoded on pD4 was suitable for use as a selectablemarker in Mortierella alpina.

In an embodiment of the present invention, vector pD4, comprising thenucleotide sequence encoding the hpt gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Mortierella alpina to reflect thecodon bias inherent in nuclear genes of Mortierella alpina in accordancewith Tables 19A-D. For each lipid biosynthesis pathway protein of Table20, the codon-optimized gene sequence can individually be operablylinked to the Mortierella alpina histone H4.1 gene promoter upstream ofthe protein-coding sequence and operably linked to the A. nidulans trpC3′UTR/terminator at the 3′ region, or downstream, of the protein-codingsequence. The transformation construct may additionally comprisehomology regions to the Mortierella alpina genome for targeted genomicintegration of the transformation vector. Homology regions may beselected to disrupt one or more genomic sites of endogenous lipidbiosynthesis pathway genes. One skilled in the art can identify suchhomology regions within the sequence of the Mortierella alpina genome(referenced in the publication by Wang et al., PLOS One, Vol. 6:12(2011)). Stable transformation of Mortierella alpina with thetransformation vector is achieved through well-known transformationtechniques including protoplast transformation or other known methods.Activity of the hpt gene product can be used as a marker to select forMortierella alpina transformed with the transformation vector on, butnot limited to, PDA medium comprising hygromycin. Growth media suitablefor Mortierella alpina lipid production include, but are not limited to,S2GYE medium, and those culture media described by Lu et al. and Ando etal. Evaluation of fatty acid profiles of Mortierella alpina lipids canbe assessed through standard lipid extraction and analytical methodsdescribed herein.

Example 33 Engineering Rhodococcus opacus PD630

Expression of recombinant genes in accordance with the present inventionin Rhodococcus opacus PD630 can be accomplished by modifying the methodsand vectors taught by Kalscheuer et al. as discussed herein. Briefly,Kalscheuer et al., Applied and Environmental Microbiology, Vol. 52(1999), pp. 508-515, reported the stable transformation of Rhodococcusopacus with plasmid DNA. Using the transformation method ofelectroporation, Kalscheuer introduced the plasmid pNC9501 intoRhodococcus opacus PD630. Plasmid pNC9501 comprised a thiostreptonresistance (thio^(r)) cassette, comprising the full nucleotide sequenceof the Streptomyces azureus 23S rRNA A1067 methyltransferase gene,including the gene's promoter and 3′ terminator sequence. Prior totransformation with pNC9501, Rhodococcus opacus was unable to propagateon medium comprising 1 mg/ml thiostrepton. Upon transformation ofRhodococcus opacus PD630 with pNC9501, transformants were obtained thatpropagated on culture medium comprising 1 mg/ml thiostrepton, therebyestablishing the use of the thiostrepton resistance cassette as aselectable marker in Rhodococcus opacus PD630. A second plasmiddescribed by Kalscheuer, pAK68, comprised the resistance thio^(r)cassette as well as the gene sequences of the Ralstonia eutrophabeta-ketothiolase (phaB), acetoacetyl-CoA reductase (phaA), andpoly3-hydroxyalkanoic acid synthase (phaC) genes forpolyhydroxyalkanoate biosynthesis, driven by the lacZ promoter. UponpAK68 transformation of a Rhodococcus opacus PD630 strain deficient inpolyhydroxyalkanoate biosynthesis, transformed Rhodococcus opacus PD630were obtained that produced higher amounts of polyhydroxyalkanoates thanthe untransformed strain. Detectable activity of the introducedRalstonia eutropha phaB, phaA, and phaC enzymes indicted that theregulatory elements encoded on the pAK68 plasmid were suitable forheterologous gene expression in Rhodococcus opacus PD630. Kalscheuerreported that selection and maintenance of the transformed Rhodococcusopacus PD630 was performed on standard Luria Broth (LB) medium, nutrientbroth (NB), or mineral salts medium (MSM) comprising thiostrepton. Othermedia and techniques used for culturing Rhodococcus opacus PD630 havebeen described (for example see Kurosawa et al., Journal ofBiotechnology, Vol. 147:3-4 (2010), pp. 212-218 and Alverez et al.,Applied Microbial and Biotechnology, Vol. 54:2 (2000), pp. 218-223).Kalscheuer reported that the transformation vectors pNC9501 and pAK68,the promoters of the Streptomyces azureus 23S rRNA A1067methyltransferase gene and lacZ gene are suitable to enable heterologousgene expression in Rhodococcus opacus PD630. In addition, Kalscheuerreported that the thio^(r) cassette encoded on pNC9501 and pAK68 wassuitable for use as a selectable marker in Rhodococcus opacus PD630.

In an embodiment of the present invention, vector pNC9501, comprisingthe nucleotide sequence encoding the thio^(r) gene product for use as aselectable marker, is constructed and modified to further comprise alipid biosynthesis pathway expression cassette sequence, therebycreating a transformation vector. The lipid biosynthesis pathwayexpression cassette encodes one or more lipid biosynthesis pathwayproteins selected from Table 20, each protein-coding sequencecodon-optimized for expression in Rhodococcus opacus PD630 to reflectthe codon bias inherent in nuclear genes of Rhodococcus opacus inaccordance with Tables 19A-D. For each lipid biosynthesis pathwayprotein of Table 20, the codon-optimized gene sequence can individuallybe operably linked to the lacZ gene promoter upstream of theprotein-coding sequence. The transformation construct may additionallycomprise homology regions to the Rhodococcus opacus PD630 genome fortargeted genomic integration of the transformation vector. Homologyregions may be selected to disrupt one or more genomic sites ofendogenous lipid biosynthesis pathway genes. One skilled in the art canidentify such homology regions within the sequence of the Rhodococcusopacus PD630 genome (referenced in the publication by Holder et al.,PLOS Genetics, Vol. 7:9 (2011). Transformation of Rhodococcus opacusPD630 with the transformation vector is achieved through well-knowntransformation techniques including electroporation or other knownmethods. Activity of the Streptomyces azureus 23S rRNA A1067methyltransferase gene product can be used as a marker to select forRhodococcus opacus PD630 transformed with the transformation vector on,but not limited to, LB medium comprising thiostrepton. Growth mediasuitable Rhodococcus opacus PD630 lipid production include, but are notlimited to those culture media discussed by Kurosawa et al. and Alvarezet al. Evaluation of fatty acid profiles of Rhodococcus opacus PD630lipids can be assessed through standard lipid extraction and analyticalmethods described herein.

Example 34 Engineering Microalgae for Fatty Acid Auxotrophy

Strain B of Example 3, Prototheca moriformis (UTEX 1435) engineered toexpress a Cuphea wrightii thioesterase (CwTE2), was used as the hostorganism for further genetic modification to knockout both endogenousthioesterase alleles, FATA1-1 and FATA1-2. Here, a first transformationconstruct was generated to integrate a neomycin expression cassette intoStrain B at the FATA1-1 locus. This construct, pSZ2226, included 5′ (SEQID NO: 30) and 3′ (SEQ ID NO: 31) homologous recombination targetingsequences (flanking the construct) to the FATA1-1 locus of the nucleargenome and a neomycin resistance protein-coding sequence under thecontrol of the C. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5)and the Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). ThisNeoR expression cassette is listed as SEQ ID NO: 15 and served as aselectable marker.

Upon transformation of pSZ2226 into Strain B, individual transformantswere selected on agar plates comprising sucrose and G418. A singleisolate, Strain H, was selected for further genetic modification. Asecond transformation construct, pSZ2236, was generated to integratepolynucleotides enabling expression of a thiamine selectable marker intoStrain H at the FATA1-2 locus. pSZ2236 included 5′ (SEQ ID NO: 32) and3′ (SEQ ID NO: 33) homologous recombination targeting sequences(flanking the construct) to the FATA1-2 genomic region for integrationinto the P. moriformis (UTEX 1435) nuclear genome and an A. thalianaTHIC protein coding region under the control of the C. protothecoidesactin promoter/5′UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This AtTHIC expression cassette is listed as SEQID NO: 23 and served as a selectable marker. Upon transformation ofStrain H with pSZ2236 to generate Strain I, individual transformants,were selected on agar plates comprising free fatty acids. Strain I wasable to propagate on agar plates and in medium lacking thiamine andsupplemented with free fatty acids.

Example 35 Engineering Microorganisms for Increased Production ofStearic Acid

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed with the plasmid construct pSZ2281 accordingto biolistic transformation methods as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. pSZ2281 included polynucleotides encoding RNAhairpins (SAD2hpC, SEQ ID NO: 34) to down-regulate the expression ofstearoyl-ACP desaturase, 5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2)homologous recombination targeting sequences (flanking the construct) tothe 6S genomic region for integration into the nuclear genome, and a S.cerevisiae suc2 sucrose invertase coding region (SEQ ID NO: 4), toexpress the protein sequence given in SEQ ID NO: 3, under the control ofC. reinhardtii β-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorellavulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiaesuc2 expression cassette is listed as SEQ ID NO: 7 and served as aselectable marker. The polynucleotide sequence encoding the SAD2hpC RNAhairpin was under the control of the C. protothecoides actinpromoter/5′UTR (SEQ ID NO: 22) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6).

Upon transformation of Strain J with construct pSZ2281, therebygenerating Strain K, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using standard fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX Strain J propagated on glucoseas a sole carbon source and three representative isolates of Strain K,propagated on sucrose as a sole carbon source, are presented in Table21.

TABLE 21 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cellsengineered to express a hairpin RNA construct targeting stearoyl ACPdesaturase gene/gene products. Area % Fatty acid Strain J Strain K-1Strain K-2 Strain K-3 Strain K-4 C8:0 0.02 C10:0 0.01 0.00 0.02 0.020.04 C12:0 0.03 0.05 0.05 0.05 0.08 C14:0 1.22 0.89 0.87 0.77 1.2 C16:026.75 29.23 28.96 27.55 28.06 C18:0 3.06 37.39 36.76 36.41 40.82 C18:159.62 23.90 24.76 26.92 22.02 C18:2 7.33 5.44 5.54 5.54 4.53 C18:3 0.14C20:0 1.43

The data presented in Table 21 show a clear impact of the expression ofSAD2 hairpin RNA construct on the C18:0 and C18:1 fatty acid profiles ofthe transformed organism. The fatty acid profiles of Strain Ktransformants comprising a SAD2 hairpin RNA construct demonstrated anincrease in the percentage of saturated C18:0 fatty acids with aconcomitant diminution of unsaturated C18:1 fatty acids. Fatty acidprofiles of the untransformed strain comprise about 3% C18:0. Fatty acidprofiles of the transformed strains comprise about 37% C18:0. These dataillustrate the successful expression and use of polynucleotides enablingexpression of a SAD RNA hairpin construct in Prototheca moriformis toalter the percentage of saturated fatty acids in the engineered hostmicrobes, and in particular in increasing the concentration of C18:0fatty acids and decreasing C18:1 fatty acids in microbial cells.

Also shown in Table 21, strain K-4 had a yet further elevated level ofstearate. Strain K4 was created by inserting the construct of strainsK1-K3 into the SAD2B locus. Thus, by knocking out one copy of the SADgene and inhibiting the remaining copies at the RNA level, a furtherreduction in oleic acid and corresponding increase in stearate wasobtained. Triglyceride analysis of RBD oil obtained from strain K4showed about 12% POP, 27% POS and 18% SOS.

Example 36 Engineering Microorganisms for Increased Production of OleicAcid Through Knockdown of an Endogenous Acyl-ACP Thioesterase

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed independently with each of the constructspSZ2402-pSZ2407 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructspSZ2402-pSZ2407 included different polynucleotides encoding a hairpinRNA targeted against Prototheca moriformis FATA1 mRNA transcripts todown-regulate the expression of fatty acyl-ACP thioesterase, 5′ (SEQ IDNO: 1) and 3′ (SEQ ID NO: 2) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the nuclear genome, and a S. cerevisiae suc2 sucroseinvertase coding region (SEQ ID NO: 4) to express the protein sequencegiven in SEQ ID NO: 3 under the control of C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette islisted as SEQ ID NO: 7 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to each hairpinare listed in Table 22. The polynucleotide sequence encoding each RNAhairpin was under the control of the C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6).

TABLE 22 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain J. Plasmid construct Hairpin designation SEQ ID NO:pSZ2402 PmFATA-hpB SEQ ID NO: 40 pSZ2403 PmFATA-hpC SEQ ID NO: 41pSZ2404 PmFATA-hpD SEQ ID NO: 42 pSZ2405 PmFATA-hpE SEQ ID NO: 43pSZ2406 PmFATA-hpF SEQ ID NO: 44 pSZ2407 PmFATA-hpG SEQ ID NO: 45

Upon independent transformation of Strain J with each of the constructslisted in Table 22, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using standard fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis (UTEX 1435) Strain J propagated onglucose as a sole carbon source and representative isolates of eachtransformation of Strain J, propagated on sucrose as a sole carbonsource, are presented in Table 23.

TABLE 23 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cellsengineered to express hairpin RNA constructs targeting fatty acyl-ACPthioesterase gene/gene products. Area % Fatty Acid Construct C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 Strain J untransformed 0 0.05 1.32 26.663.1 59.07 7.39 PmFATA-hpB 0.04 0.07 1.36 24.88 2.24 61.92 6.84 0 0.081.33 25.34 2.39 61.72 6.5 0 0.07 1.29 25.44 2.26 61.7 6.69 0 0.06 1.3325.1 2.37 61.56 6.87 PmFATA-hpC 0 0.08 1.18 22.03 1.71 63.8 8.63 0 0.071.21 24.5 2.23 62.32 7.19 0 0.08 1.29 24.93 2.24 62.02 7.01 0.05 0.061.29 25.45 2.26 61.81 6.76 PmFATA-hpD 0 0.02 0.68 15.8 1.88 72.64 6.96 00.03 0.78 17.56 1.7 71.8 6.03 0 0.03 0.92 19.04 2.03 68.82 7.05 0 0.041.27 23.14 2.25 65.27 6.07 PmFATA-hpE 0 0.03 0.79 18.55 2.13 69.66 6.770 0.04 1.11 21.01 1.74 65.18 8.55 0 0.03 1.08 21.11 1.54 64.76 8.87 00.03 1.17 21.93 1.71 63.89 8.77 PmFATA-hpF 0.03 0.04 0.34 8.6 1.69 78.088.87 0 0.03 0.49 10.2 1.52 76.97 8.78 0 0.03 1 20.47 2.22 66.34 7.45 00.03 1.03 21.61 1.88 65.39 7.76 PmFATA-hpG 0 0.03 1.03 20.57 2.36 64.738.75 0 0.03 1.2 24.39 2.47 61.9 7.49 0 0.04 1.29 24.14 2.29 61.41 8.22

The data presented in Table 23 show a clear impact of the expression ofFATA hairpin RNA constructs on the C18:0 and C18:1 fatty acid profilesof the transformed organism. The fatty acid profiles of Strain Jtransformants comprising a FATA hairpin RNA construct demonstrated anincrease in the percentage of C18:1 fatty acids with a concomitantdiminution of C16:0 and C18:0 fatty acids. Fatty acid profiles of theuntransformed Strain J are about 26.66% C16:0, 3% C18:0, and about 59%C18:1 fatty acids. In contrast, the fatty acid profiles of thetransformed strains comprise as low as 8.6% C16:0 and 1.54% C18:0 andgreater than 78% C18:1 fatty acids.

These data illustrate the utility and successful use of polynucleotideFATA RNA hairpin constructs in Prototheca moriformis to alter the fattyacids profile of engineered microbes, and in particular in increasingthe concentration of C18:1 fatty acids and decreasing C18:0 and C16:0fatty acids in microbial cells.

Example 37 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI or KASIV Overexpression

This example describes the use of recombinant polynucleotides thatencode KASI or KASIV enzymes to engineer microorganisms in which thefatty acid profiles of the transformed microorganisms have been enrichedin lauric acid, C10:0, and total saturated fatty acids.

Each of the constructs pSZD1132, pSZD1133, pSZD1134, or pSZD1201 wasused independently to transform Strain B of Example 3, Protothecamoriformis (UTEX 1435) engineered to express a Cuphea wrightiithioesterase (CwTE2), according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the above constructsincluded different polynucleotides encoding a KASI or KASIV enzyme, 5′(SEQ ID NO: 13) and 3′ (SEQ ID NO: 14) homologous recombinationtargeting sequences (flanking the construct) to the pLoop genomic regionfor integration into the nuclear genome, and a neomycin resistanceprotein-coding sequence under the control of the C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 5) and the Chlorella vulgarisnitrate reductase 3′ UTR (SEQ ID NO: 6). This NeoR expression cassetteis listed as SEQ ID NO: 15 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to eachconstruct are listed in Table 20. The polynucleotide sequence encodingeach KAS enzyme was under the control of the P. moriformis UTEX 1435Amt03 promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′UTR (SEQ ID NO: 6). The protein coding regions of the KAS enzymes andneomycin resistance gene were codon optimized to reflect the codon biasinherent in P. moriformis UTEX 1435 nuclear genes as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon transformation of individual plasmids into Strain B, positiveclones were selected on agar plates comprising G418. Individualtransformants were clonally purified and grown on sucrose as a solecarbon source at pH 7.0 under conditions suitable for lipid productionas detailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using standard fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of Strain B and four positive transformants of eachof pSZ2046 (Strains M-P, 1-4) are presented in Table 24.

TABLE 24 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain B. Plasmid KASI/KASIV construct source Transitpeptide SEQ ID NO: pSZD1134 Cuphea wrightii Native SEQ ID NO: 46 GenBankAccession No. U67317 pSZD1201 Cuphea wrightii PmSAD SEQ ID NO: 47pSZD1132 Cuphea Native SEQ ID NO: 48 pulcherrima GenBank Accession No.AAC68860 pSZD1133 Cuphea hookeriana Native SEQ ID NO: 49

TABLE 25 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainBengineered for increased C10, lauric acid, and total saturated fattyacids. Fatty Acid (Area %) Plasmid C10- % Saturates/ construct(s) No.C10 C12 C14 C16 C18:0 C18:1 C18:2 C12  Total pSZ1283 7.89 35.49 16.5811.5 1.09 19.64 6.49 43.38 72.55 pSZ1283, 1 14.94 43.97 12.19 7.56 0.7214.11 5.31 58.91 79.38 pSZD1134 pSZ1283, 2 10.27 39.61 15.35 9.61 0.9417.1 5.88 49.88 75.78 pSZD1134 pSZ1283, 3 11.69 41.83 15.21 8.77 0.8315.04 5.40 53.52 78.33 pSZD1134 D1134-20 4 10.76 40.77 15.32 9.19 0.8816.06 5.76 51.53 76.92 pSZ1283, 1 10.77 40.31 15.21 9.43 0.88 16.18 5.9751.08 76.6 pSZD1132 pSZ1283, 2 9.19 37.03 15.02 10.52 1.00 19.63 6.2946.22 72.76 pSZD1132 pSZ1283, 3 8.97 36.09 15.01 10.77 1.05 20.38 6.3945.06 71.89 pSZD1132 pSZ1283, 4 9.51 38.12 14.96 9.96 0.94 18.93 6.3247.63 73.49 pSZD1132 pSZ1283, 1 13.06 46.21 9.84 7.12 0.75 16.7 5.2259.27 76.98 pSZD1201 pSZ1283, 2 11.02 43.91 13.01 7.78 0.86 16.53 5.7754.93 76.58 pSZD1201 pSZ1283, 3 11.59 45.14 12.41 7.61 0.82 15.72 5.6556.73 77.57 pSZD1201 pSZ1283, 4 10.66 41.32 13.74 8.75 0.68 18.64 5.2151.98 75.15 pSZD1201 pSZ1283, 1 6.90 36.08 15.15 11.02 1.00 21.74 6.7742.98 70.15 pSZD1133 pSZ1283, 2 7.01 35.88 15.01 10.75 1.07 22.02 6.9342.89 69.72 pSZD1133 pSZ1283, 3 10.65 41.94 12.38 8.48 0.85 18.28 6.1552.59 74.3 pSZD1133 pSZ1283, 4 10.23 41.88 12.58 8.52 0.82 18.48 6.2252.11 74.03 pSZD1133

The data presented in Table 25 show a clear impact of the exogenousexpression of KASI and KASIV enzymes on the C10:0 and C12 fatty acidprofiles of the transformed organism. The fatty acid profiles of StrainB, expressing the Cuphea wrightii thioesterase alone, comprised about 8%C10:0 and about 35.5% C12:0, with saturated fatty acids accounting for72.55% of total fatty acids. In contrast, transformants of Strain Bengineered to additionally express a Cuphea wrightii KASI with a P.moriformis stearoyl ACP desaturase transit peptide were characterized bya fatty acid profile of about 13% C10:0 and about 46% C12:0. Saturatedfatty acids accounted for as high as 77% in transformants of Strain Bco-expressing the C. wrightii KASI fusion protein. Similarly,transformants of Strain B engineered to express the C. wrightii KASIwith the enzyme's native transit peptide were characterized by a fattyacid profile of about 15% C10, about 44% C12, and about 79% saturatedfatty acids. The fatty acid profiles or many transformants of Strain Bexpressing either Cuphea pulcherrima KASIV or Cuphea hookeriana KASIValso displayed elevated C10% and C12% levels, compared to the fatty acidprofile of Strain B itself.

These data demonstrate the utility and effectiveness of polynucleotidesenabling expression of KASI and KASIV constructs in Protothecamoriformis (UTEX 1435) to alter the percentage of saturated fatty acidsin the engineered host microbes, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids in microbial cells.

Example 38 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI Knockout

This example describes the use of recombinant polynucleotides thatdisrupt different KASI alleles to engineer microorganisms in which thefatty acid profiles of the transformed microorganisms have been enrichedin C10:0 and midchain fatty acids.

Constructs pSZ2302 and pSZ2304 were used to independently transformStrain B of Example 3, Prototheca moriformis (UTEX 1435) engineered toexpress a Cuphea wrightii thioesterase (CwTE2), according to biolistictransformation methods as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. pSZ2302 included 5′ (SEQ ID NO: 50) and 3′ (SEQ IDNO: 51) homologous recombination targeting sequences (flanking theconstruct) to the KAS1 allele 1 genomic region for integration into theP. moriformis nuclear genome, an A. thaliana THIC protein coding regionunder the control of the C. protothecoides actin promoter/5′UTR (SEQ IDNO: 22) and C. vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). pSZ2304included 5′ (SEQ ID NO: 52) and 3′ (SEQ ID NO: 53) homologousrecombination targeting sequences (flanking the construct) to the KAS1allele 2 genomic region for integration into the P. moriformis nucleargenome, an A. thaliana THIC protein coding region under the control ofthe C. protothecoides actin promoter/5′UTR (SEQ ID NO: 22) and C.vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). This AtTHIC expressioncassette is listed as SEQ ID NO: 23 and served as a selection marker.The protein coding region of AtTHIC was codon optimized to reflect thecodon bias inherent in P. moriformis UTEX 1435 nuclear genes asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon independent transformation pSZ2302 and pSZ2304 into Strain B,thereby generating Strain Q and R, positive clones were selected on agarplates comprising thiamine. Individual transformants were clonallypurified and cultivated on sucrose as a sole carbon source at pH 5.0 orpH 7.0 under heterotrophic conditions suitable for lipid production asdetailed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples were preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples were analyzed using fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. The fatty acid profiles (expressed as Area % oftotal fatty acids) of Strain B and positive pSZ2302 (Strain Q, 1-5) andpSZ2304 (Strain R, 1-5) transformants are presented in Tables 26 and 27.

TABLE 26 Fatty acid profiles of Prototheca moriformis (UTEX 1435)Strains B, Q, and R engineered for increased midchain fatty acids,cultured at pH 5.0. Transformation Fatty Acid (Area %) Strain plasmid(s)C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C10-C14 UTEX 1435 None 0.000.04 1.28 26.67 3.05 59.96 7.19 1.32 Strain B pSZ1283 0.01 0.09 1.0921.60 2.21 65.15 7.94 1.19 Strain Q-1 pSZ1283, 0.08 1.21 7.52 38.71 1.3838.32 8.75 8.81 pSZ2302 Strain Q-2 pSZ1283, 0.15 1.36 7.51 38.23 1.3338.27 8.94 9.02 pSZ2302 Strain Q-3 pSZ1283, 0.16 1.43 7.49 38.88 1.3037.58 8.73 9.08 pSZ2302 Strain Q-4 pSZ1283, 0.00 1.71 7.42 37.67 1.4337.26 10.38 9.13 pSZ2302 Strain Q-5 pSZ1283, 0.13 1.21 7.36 38.81 1.3138.07 8.71 8.7 pSZ2302 Strain R-1 pSZ1283, 0.19 1.78 8.47 40.11 1.3433.46 9.98 10.44 pSZ2304 Strain R-2 pSZ1283, 0.90 8.00 7.78 28.96 1.1530.26 17.14 16.68 pSZ2304 Strain R-3 pSZ1283, 0.26 3.58 7.77 34.98 1.5632.86 14.60 11.61 pSZ2304 Strain R-4 pSZ1283, 1.64 13.50 7.61 21.38 0.9036.13 14.73 22.75 pSZ2304 Strain R-5 pSZ1283, 1.03 9.63 7.56 25.61 1.0031.70 18.23 18.22 pSZ2304

TABLE 27 Fatty acid profiles of Prototheca moriformis (UTEX 1435),Strains B, Q, and R engineered for increased midchain fatty acids,cultured at pH 7.0. Transformation Fatty Acid (Area %) Strain plasmid(s)C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C10-C14 UTEX None 0.01 0.041.34 27.94 3.24 57.46 7.88 1.39 1435 Strain B pSZ1283 4.72 29.57 15.5612.63 1.20 27.65 7.39 49.85 Strain Q-1 pSZ1283, pSZ2302 16.00 50.61 9.525.33 0.54 11.79 5.28 76.13 Strain Q-2 pSZ1283, pSZ2302 16.32 49.79 9.825.52 0.54 12.28 4.87 75.93 Strain Q-3 pSZ1283, pSZ2302 15.08 47.58 10.235.93 0.56 15.12 4.50 72.89 Strain Q-4 pSZ1283, pSZ2302 14.27 47.30 10.446.17 0.56 15.50 4.59 72.01 Strain Q-5 pSZ1283, pSZ2302 14.75 47.28 10.326.04 0.59 15.50 4.65 72.35 Strain R-1 pSZ1283, pSZ2304 21.25 55.42 7.973.65 0.00 5.46 5.66 84.64 Strain R-2 pSZ1283, pSZ2304 13.00 55.05 10.885.78 0.28 7.90 6.29 78.93 Strain R-3 pSZ1283, pSZ2304 12.89 53.15 11.116.13 0.00 9.87 6.13 77.15 Strain R-4 pSZ1283, pSZ2304 12.80 51.64 13.866.69 0.00 7.51 6.70 78.3 Strain R-5 pSZ1283, pSZ2304 16.61 51.42 9.845.27 0.33 11.15 4.79 77.87

The data presented in Tables 26 and 27 show a clear impact of disruptionof different KASI alleles on the fatty acid profiles of the transformedorganisms. When cultivated at pH 5.0, the fatty acid profiles ofPrototheca moriformis (UTEX 1435) and Prototheca moriformis (UTEX 1435)Strain B, expressing a Cuphea wrightii FATB2 thioesterase under controlof a pH regulatable promoter were very similar. These profiles werecharacterized by about 1% C14:0, about 21-26% C16:0, about 2-3% C18:0,about 60-65% C18:1, about 7% C18:2, with C10-C14 fatty acids comprisingabout 1.19-1.3% of total fatty acids. In contrast, when cultivated at pH5.0, Strain B further engineered to disrupt KASI allele 1 (Strain Q) orKASI allele 2 (Strain R) demonstrated altered fatty acid profiles thatwere characterized by increased levels of C12, increased levels of C14,decreased levels of C18, and decreased levels of C18:1 fatty acidscompared to Strain B or UTEX 1435. The fatty acid profiles of isolatesof Strains Q and R differed in that Strain R (allele 2 knockout)isolates had generally greater C12s and lower C16s and C18:1s thanStrain Q (allele 1 knockout).

When cultivated at pH 7.0, the fatty acid profile of Protothecamoriformis (UTEX 1435) is distinct from that Prototheca moriformis (UTEX1435) Strain B expressing a Cuphea wrightii FATB2 thioesterase undercontrol of a pH regulatable promoter. When cultured at pH 7.0, Strain Bwas characterized by a fatty acid profile elevated in C10, C12, and C14fatty acids (these comprised about 50% of the total fatty acids). Whencultured at pH 7.0, Strain Q and Strain R demonstrated fatty acidprofiles with still further increases in C10, C12, and C14 fatty acidsand still further decreases in C18:0 and C18:1 fatty acids relative tothat of Strain B. Again, differences in fatty acid profiles betweenStrain Q and R were observed with the profile of Strain R comprisinggreater percentage levels of C12 and lower levels of C18:1 than that ofStrain Q.

These data illustrate the successful expression and use ofpolynucleotides enabling expression of KASI and KASIV constructs inPrototheca moriformis to alter the percentage of saturated fatty acidsin the engineered host microbes, and in particular in increasing theconcentration of C10:0 and C12:0 fatty acids and decreasing theconcentration of C18:0 and C18:1 fatty acids in microbial cells. Inaddition, the data here indicate the different KASI alleles can bedisrupted to result in altered fatty acid profiles of the transformedorganisms.

Example 39 Engineering Microorganisms for Increased Production ofMid-Chain Fatty Acids Through KASI Knockdown

This example describes the use of recombinant polynucleotides thatencode RNA hairpins to attenuate a KASI enzyme to engineer amicroorganism in which the fatty acid profile of the transformedmicroorganism has been enriched in midchain fatty acids.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain S, was transformed independently with each of the constructspSZ2482-pSZ2485 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructspSZ2482-pSZ2485 included different polynucleotides encoding hairpin RNAstargeted against Prototheca moriformis (UTEX 1435) KASI mRNA transcriptsto down-regulate the expression of fatty acyl-ACP thioesterase, 5′ (SEQID NO: 1) and 3′ (SEQ ID NO: 2) homologous recombination targetingsequences (flanking the construct) to the 6S genomic region forintegration into the nuclear genome, and a S. cerevisiae suc2 sucroseinvertase coding region (SEQ ID NO: 4) to express the protein sequencegiven in SEQ ID NO: 3 under the control of C. reinhardtii β-tubulinpromoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitrate reductase3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expression cassette islisted as SEQ ID NO: 7 and served as a selectable marker. Sequencelisting identities for the polynucleotides corresponding to each KASIhairpin are listed in Table 28. The polynucleotide sequence encodingeach RNA hairpin was under the control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6). The protein coding region of the suc2 expressioncassette was codon optimized to reflect the codon bias inherent in P.moriformis UTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

TABLE 28 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain S. Transformation construct Hairpin SEQ ID NO:pSZ2482 KASI hairpin B SEQ ID NO: 54 pSZ2483 KASI hairpin C SEQ ID NO:55 pSZ2484 KASI hairpin D SEQ ID NO: 56 pSZ2485 KASI hairpin E SEQ IDNO: 57

Upon independent transformation of Strain S with each of the constructslisted in Table 28, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 propagated on glucose as asole carbon source and four representative isolates of eachtransformation of Strain S, propagated on sucrose as a sole carbonsource, are presented in Table 29.

TABLE 29 Fatty acid profiles of Prototheca moriformis (UTEX 1435) cellsengineered to express hairpin RNA constructs targeting KASI gene/geneproducts. Fatty Acid (Area %) Strain Plasmid Number C10:0 C12:0 C14:0C16:0 C18:0 C18:1 C18:2 C18:3 UTEX 1435 none 1 0.00 0.04 1.45 27.97 3.1858.35 6.78 0.60 Stain S psZ2482 1 0.19 0.74 8.47 38.30 2.15 36.24 9.451.42 2 0.07 0.25 4.16 32.46 2.62 49.57 7.73 0.82 3 0.03 0.10 2.68 27.482.65 56.40 8.14 0.55 4 0.03 0.10 2.60 27.44 2.01 55.54 9.15 0.78 pSZ24831 0.00 0.06 1.94 30.58 1.55 53.26 9.31 0.76 2 0.20 0.05 1.76 28.01 2.3156.61 8.70 0.60 3 0.00 0.06 1.60 24.38 2.65 58.25 9.93 1.15 4 0.00 0.041.56 26.65 2.96 60.06 6.92 0.52 pSZ2484 1 0.72 3.71 19.15 38.03 1.6814.22 15.00 4.21 2 0.66 2.76 16.34 38.19 1.78 18.52 14.91 3.38 3 0.692.96 16.20 37.28 1.77 19.05 15.26 3.48 4 0.18 0.70 8.61 36.80 2.35 36.2210.89 1.10 pSZ2485 1 0.00 0.04 1.41 25.34 3.16 60.12 7.78 0.48 2 0.030.04 1.41 23.85 2.19 61.23 8.75 0.67 3 0.00 0.04 1.41 24.41 2.23 60.648.69 0.67 4 0.00 0.04 1.41 24.51 2.16 60.85 8.91 0.66

The data presented in Table 29 show a clear impact of the expression ofKAS hairpin RNA constructs on the fatty acid profiles of the transformedorganisms. The fatty acid profiles of Strain S transformants comprisingeither pSZ2482 or pSZ2484 KASI hairpin RNA construct demonstrated anincrease in the percentage of C10, C12, C14, and C16 fatty acids with aconcomitant diminution of C18:0 and C18:1 fatty acids relative to thefatty acid profile of UTEX 1435.

These data illustrate the utility and successful use of polynucleotideKASI RNA hairpin constructs in Prototheca moriformis (UTEX 1435) toalter the fatty acids profile of engineered microbes, and in particularin increasing the concentration of midchain fatty acids and decreasingC18:0 and C18:1 fatty acids in microbial cells.

Example 40 Engineering Microorganisms for Increased Production ofStearic Acid Through Elongase Overexpression

This example describes the use of recombinant polynucleotides thatencode elongases to engineer a microorganism in which the fatty acidprofile of the transformed microorganism has been enriched in stearicacid, arachidic acid, and docosadienoic acid.

A classically mutagenized strain of Prototheca moriformis (UTEX 1435),Strain J, was transformed independently with each of the constructspSZ2323, pSZ2324, or pSZ2328 according to biolistic transformationmethods as described in PCT/US2009/066141, PCT/US2009/066142,PCT/US2011/038463, PCT/US2011/038464, and PCT/US2012/023696. Each of theconstructs included a protein coding region to overexpress an elongase,5′ (SEQ ID NO: 1) and 3′ (SEQ ID NO: 2) homologous recombinationtargeting sequences (flanking the construct) to the 6S genomic regionfor integration into the nuclear genome, and a S. cerevisiae suc2sucrose invertase coding region (SEQ ID NO: 4) to express the proteinsequence given in SEQ ID NO: 3 under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQ ID NO: 5) and Chlorella vulgaris nitratereductase 3′ UTR (SEQ ID NO: 6). This S. cerevisiae suc2 expressioncassette is listed as SEQ ID NO: 7 and served as a selectable marker.Sequence listing identities for the polynucleotides corresponding toeach elongase are listed in Table 30. The polynucleotide sequenceencoding each elongase was under control of the P. moriformis Amt03promoter/5′UTR (SEQ ID NO: 8) and C. vulgaris nitrate reductase 3′ UTR(SEQ ID NO: 6). The protein coding regions of the exogenous elongasesand the suc2 expression cassette were codon optimized to reflect thecodon bias inherent in P. moriformis UTEX 1435 nuclear genes asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

TABLE 30 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain J. GenBank SEQ Plasmid construct Elongase sourceAccession No. ID NO: pSZ2328 Marchantia polymorpha AAP74370 58, 59pSZ2324 Trypanosoma brucei AAX70673 60, 61 pSZ2323 Saccharomycescerevisiae P39540 62, 63

Upon independent transformation of Strain J with the constructs listedin Table 30, positive clones were selected on agar plates containingsucrose as a sole carbon source. Individual transformants were clonallypurified and propagated under heterotrophic conditions suitable forlipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 Strain J propagated onglucose as a sole carbon source and three representative isolates ofeach transformation of Strain J, propagated on sucrose as a sole carbonsource are presented in Table 31.

TABLE 31 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainJ cells engineered to overexpress elongases. Plasmid Fatty Acid Area %construct No C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3α C20:0 C22:2n6None 1 1.39 27.42 0.77 3.33 57.46 8.05 0.61 0.30 0.03 pSZ2328 1 1.2519.23 0.85 8.26 57.54 9.34 0.79 0.73 0.94 pSZ2328 2 1.22 17.76 0.69 7.8660.56 9.38 0.59 0.6 0.47 pSZ2328 3 1.26 18.37 0.92 7.83 58.77 10.01 0.720.64 0.52 pSZ2324 1 1.51 22.97 1.09 8.71 53.01 9.63 0.65 0.68 0.55pSZ2324 2 1.29 20.6 0.92 7.53 56.97 9.92 0.73 0.64 0.43 pSZ2324 3 1.2820.59 0.93 7.33 57.52 9.68 0.65 0.58 0.42 pSZ2323 1 1.65 27.27 0.67 3.5656.68 8.72 0.33 0.36 0.00 pSZ2323 2 1.56 28.44 0.74 3.36 55.22 9.07 0.460.39 0.03 pSZ2323 3 1.64 28.7 0.75 3.34 55.29 8.59 0.49 0.36 0.02

The data presented in Table 31 show a clear impact of the expression ofMarchantia polymorpha and Trypanosoma brucei enzymes on the C14, C16,C18:0, C20:0, and C22:2n6 fatty acid profiles of the transformedorganisms. The fatty acid profile of untransformed Strain J was about27.42% C16:0, about 3% C18:0, about 57.5% C18:1, about 0.3% C20:0 andabout 0.03% C22:2n6 fatty acids. In contrast to that of Strain J, thefatty acid profiles of Strain J transformed with different plasmidconstructs to express elongases comprised lower percentage levels of C16and higher percentage levels of C18:0, C20:0, and C22:2n6 fatty acids.The result of overexpression of Marchantia polymorpha elongase was abouta 2.5 fold increase in percentage levels of C18:0 fatty acids, a 2 foldincrease in percentage levels of C20:0 fatty acids, and about a 15 to 30fold increase in percentage levels of C22:2n6 fatty acids relative tothe fatty acid profile of Strain J.

These data illustrate the successful use of polynucleotides encodingelongases for expression in Prototheca moriformis (UTEX 1435) to alterthe fatty acid profile of engineered microbes, and in particular inincreasing the concentration of C18:0, C20:0, and C22:2n6 fatty acidsand decreasing C16:0 fatty acids in recombinant microbial cells.

Example 41 Engineering Microorganisms for Increased Production ofStearic Acid Through Acyl-ACP Thioesterase Overexpression

This example describes the use of recombinant polynucleotides thatencode different C18:0-preferring acyl-ACP thioesterases to engineermicroorganisms in which the fatty acid profiles of the transformedmicroorganisms have been enriched in stearic acid.

Classically mutagenized strains of Prototheca moriformis (UTEX 1435),Strain J or Strain A, were transformed independently with the constructslisted in Table 32 according to biolistic transformation methods asdescribed in PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Each of the constructsincluded a protein coding region to overexpress a fatty acyl-ACPthioesterase with a C-terminal 3×FLAG® epitope tag, 5′ (SEQ ID NO: 1)and 3′ (SEQ ID NO: 2) homologous recombination targeting sequences(flanking the construct) to the 6S genomic region for integration intothe nuclear genome, and a S. cerevisiae suc2 sucrose invertase codingregion (SEQ ID NO: 4) to express the protein sequence given in SEQ IDNO: 3 under the control of C. reinhardtiiβ-tubulin promoter/5′UTR (SEQID NO: 5) and Chlorella vulgaris nitrate reductase 3′ UTR (SEQ ID NO:6). This S. cerevisiae suc2 expression cassette is listed as SEQ ID NO:7 and served as a selectable marker. Sequence listing identities for thepolynucleotides corresponding to each thioesterase are listed in Table32. The polynucleotide sequence encoding each thioesterase was undercontrol of the P. moriformis Amt03 promoter/5′UTR (SEQ ID NO: 8) and C.vulgaris nitrate reductase 3′ UTR (SEQ ID NO: 6). The protein codingregions of the exogenous thioesterases and the suc2 expression cassettewere codon optimized to reflect the codon bias inherent in P. moriformisUTEX 1435 nuclear genes as described in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696.

TABLE 32 Plasmid constructs used to transform Prototheca moriformis(UTEX 1435) Strain A or Strain J. Acyl-ACP Thioesterase, Acyl-ACPTransit Plasmid GenBank Thioesterase Peptide SEQ ID construct AccessionNo. source source NO: pSZD581 FATA, CAA52070 Brassica napus native 64,65 pSZD643 FATA, CAA52070 Brassica napus UTEX 66, 67 250 SAD pSZD645FATA, AAA33019 C. tinctorius UTEX 68, 69 250 SAD pSZD644 FATA, ABS30422Ricinis communis native 70, 71 pSZD1323 FATA, AAB51523 G. mangostananative 72, 73 pSZD1320 FATA Theobroma native 74, 75 cacao

Upon independent transformation of Strain A or J with the constructslisted in Table 32, positive clones were selected on agar platescontaining sucrose as a sole carbon source. Individual transformantswere clonally purified and propagated under heterotrophic conditionssuitable for lipid production as those detailed in PCT/US2009/066141,PCT/US2009/066142, PCT/US2011/038463, PCT/US2011/038464, andPCT/US2012/023696. Lipid samples were prepared from dried biomass andanalyzed using fatty acid methyl ester gas chromatography flameionization detection methods as described in Example 1 (also seePCT/US2012/023696). The fatty acid profiles (expressed as Area % oftotal fatty acids) of P. moriformis UTEX 1435 Strain J propagated onglucose as a sole carbon source and representative isolates of eachtransformation of Strain J, propagated on sucrose as a sole carbonsource are presented in Table 33.

TABLE 33 Fatty acid profiles of Prototheca moriformis (UTEX 1435) StrainJ cells engineered to overexpress exogenous acyl-ACP thioesteraseenzymes. Fatty Acid Area % Plasmid C18: Strain construct No. C14:0 C16:0C18:0 C18:1 C18:2 3α A None 1 1.08 25.48 3.23 59.70 8.25 0.70 J None 11.41 27.33 3.38 57.07 8.15 0.64 A pSZD581 1 1.02 26.60 14.47 44.80 10.050.65 2 1.08 28.24 13.57 43.89 10.07 0.68 3 0.97 24.70 9.13 50.85 11.270.82 A pSZD643 1 1.39 26.97 16.21 44.10 8.43 0.83 2 1.37 27.91 11.1548.31 8.40 0.78 A pSZD645 1 0.90 23.39 8.35 50.69 13.34 0.96 A pSZD644 11.67 19.70 4.40 59.15 12.32 1.01 J pSZD1323 1 1.33 23.26 9.28 53.4210.35 0.69 2 1.47 26.84 7.36 52.78 9.29 0.64 3 1.43 26.31 6.05 54.459.37 0.66 J pSZD1320 1 1.30 24.76 3.84 60.90 6.96 0.55 2 1.36 26.30 3.2758.19 8.66 0.48 3 1.39 25.51 3.18 58.78 8.85 0.45

The data presented in Table 33 show a clear impact of the expression ofexogenous acyl-ACP enzymes on the fatty acid profiles of the transformedmicroorganisms. The fatty acid profiles of untransformed Strain A and Jwere about 25% C16:0, about 3.3% C18:0, about 57 to 60% C18:1. Incontrast, the fatty acid profiles of Strain A transformed with differentplasmid constructs to express acyl-ACP enzymes comprised greaterpercentage levels of C18:0 and lower percentage levels of C18:1 fattyacids than that of Strain A. Expression of FATA enzymes from B. napus,C. tinctorius, R. communis and G. mangostana in Strain A or J enabledthe accumulation of stearate levels in the transformed organisms. Theresult of overexpression of a Brassica napus acyl-ACP thioestearse wasabout a 2 to 5 fold increase in the percentage levels of C18:0 fattyacids of the fatty acid profile of the transformed organsisms relativeto the fatty acid profile of Strain A. Fatty acid profiles of cellsengineered to overexpress a G. mangostana acyl-ACP FATA thioesterasewith a C. protothecoides SAD 1 transit peptide were characterized byabout a 2 to 3 fold increase in the percentage levels of C18:0 fattyacids of the fatty acid profile of the transformed organism relative tothe fatty acid profile of Strain J.

These data illustrate the utility and effective use of polynucleotidesencoding fatty acyl-ACP thioesterases for expression in Protothecamoriformis (UTEX 1435) to alter the fatty acid profile of engineeredmicrobes, and in particular in increasing the concentration of C18:0 anddecreasing C18:1 fatty acids in recombinant microbial cells.

Example 42 Engineering Microorganisms for Increased Production of ErucicAcid Through Elongase or Beta-Ketoacyl-CoA Synthase Overexpression

In an embodiment of the present invention, a recombinant polynucleotidetransformation vector operable to express an exogenous elongase orbeta-ketoacyl-CoA synthase in an optionally plastidic oleaginous microbeis constructed and employed to transform Prototheca moriformis (UTEX1435) according to the biolistic transformation methods as described inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696 to obtain a cell increased forproduction of erucic acid. The transformation vector includes a proteincoding region to overexpress an elongase or beta-ketoacyl-CoA synthasesuch as those listed in Table 5, promoter and 3′UTR control sequences toregulate expression of the exogenous gene, 5′ and 3′ homologousrecombination targeting sequences targeting the recombinantpolynucleotides for integration into the P. moriformis (UTEX 1435)nuclear genome, and nucleotides operable to express a selectable marker.The protein-coding sequences of the transformation vector arecodon-optimized for expression in P. moriformis (UTEX 1435) as describedin PCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Recombinant polynucleotidesencoding promoters, 3′ UTRs, and selectable markers operable forexpression in P. moriformis (UTEX 1435) are disclosed herein and inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696.

Upon transformation of the transformation vector into P. moriformis(UTEX 1435) or a classically-mutagenized strain of P. moriformis (UTEX1435), positive clones are selected on agar plates. Individualtransformants are clonally purified and cultivated under heterotrophicconditions suitable for lipid production as detailed inPCT/US2009/066141, PCT/US2009/066142, PCT/US2011/038463,PCT/US2011/038464, and PCT/US2012/023696. Lipid samples are preparedfrom dried biomass from each transformant and fatty acid profiles fromthese samples are analyzed using fatty acid methyl ester gaschromatography flame ionization (FAME GC/FID) detection methods asdescribed in Example 1. As a result of these manipulations, the cell mayexhibit an increase in erucic acid of at least 5, 10, 15, or 20 fold.

Example 43 Generation of Capric, Lauric, and Myristic Acid Rich Oils inStrain UTEX1435 by the Expression of Cuphea PSR23 LPAATs

We tested the effect of expression of two 1-acyl-sn-glycerol-3-phosphateacyltransferases (LPAATs) in a previously described P. moriformis (UTEX1435) transgenic strain, expressing the acyl ACP thioesterase (FATB2)from Cuphea wrightii. The LPAAT2 and LPAAT3 genes from Cuphea PSR23(CuPSR23) were identified by analysis of a combination of CuPSR23genomic sequences and transcriptomic sequences derived from seed RNAs.The two LPAATs have not been previously described. The genes were codonoptimized to reflect UTEX 1435 codon usage. Transformations, cellculture, lipid production and fatty acid analysis were all carried outas previously described.

Increased Capric, Lauric, and Myristic Accumulation in Strain B by theExpression of the Cuphea PSR231-acyl-sn-glycerol-3-phosphateacyltransferases (LPAAT2 and LPAAT3) [pSZ2299 and pSZ2300,Respectively]:

In this example, transgenic strains were generated via transformation ofstrain B with the constructs pSZ2299 or pSZ2300, encoding CuPSR23 LPAAT2and LPAAT3, respectively. The transgenic strains were selected forresistance to the antibiotic G418. Construct pSZ2299 can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT2-1:CvNR::pLOOP3′.Construct pSZ2300 can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAAT3-1:CvNR::pLOOP3′. Thesequence of the transforming DNA (pSZ2299 and pSZ2300) is providedbelow. The relevant restriction sites in the construct from 5′-3′,BspQI, KpnI, XbaI, Mfe I, BamHI, EcoRI, SpeI, XhoI, SacI, BspQI,respectively, are indicated in lowercase, bold, and underlined. BspQIsites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences at the 5′ and 3′ end of the construct representgenomic DNA from UTEX 1435 that target integration to the pLoop locusvia homologous recombination. Proceeding in the 5′ to 3′ direction, theselection cassette has the C. reinhardtii β-tubulin promoter drivingexpression of the NeoR gene (conferring resistance to G418) and theChlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. The promoter isindicated by lowercase, boxed text. The initiator ATG and terminator TGAfor NeoR are indicated by uppercase italics, while the coding region isindicated with lowercase italics. The 3′ UTR is indicated by lowercaseunderlined text. The spacer region between the two cassettes isindicated by upper case text. The second cassette containing the codonoptimized LPAAT2 gene (pSZ2299) or LPAAT3 gene (pSZ2300) from CupheaPSR23 is driven by the Prototheca moriformis endogenous AMT3 promoter,and has the same Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR.In this cassette, the AMT3 promoter in indicated by lowercase, boxedtext. The initiator ATG and terminator TGA for the CuPSR23 LPAAT2 andLPAAT3 genes are indicated in uppercase italics, while the codingregions are indicated by lowercase italics. The 3′ UTR is indicated bylowercase underlined text. The final constructs were sequenced to ensurecorrect reading frames and targeting sequences.

pSZ2299 Transforming Construct gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatctcgaaccccacttgcactctgcaatggtatgggaaccacggggcagtcttgtgtgggtcgcgcctatcgcggtcggcgaagaccgggaag

aggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc CGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTC

ttcgcctccggcctgatcatcaacctgttccaggcgctgtgcttcgtcctgatccgccccctgtccaagaacgcctaccgccgcatcaaccgcgtgttcgcggagctgctgctgtccgagctgctgtgcctgttcgactggtgggcgggcgcgaagctgaagctgttcaccgaccccgagacgttccgcctgatgggcaaggagcacgccctggtcatcatcaaccacatgaccgagctggactggatggtgggctgggtgatgggccagcacttcggctgcctgggctccatcatctccgtcgccaagaagtccacgaagttcctgcccgtgctgggctggtccatgtggttctccgagtacctgtacctggagcgctcctgggccaaggacaagtccaccctgaagtcccacatcgagcgcctgatcgactaccccctgcccttctggctggtcatcttcgtcgagggcacccgcttcacgcgcacgaagctgctggcggcccagcagtacgcggtctcctccggcctgcccgtcccccgcaacgtcctgatcccccgcacgaaggcttcgtctcctgcgtgtcccacatgcgctccttcgtccccgcggtgtacgacgtcacggtggcgttccccaagacgtcccccccccccacgctgctgaacctgttcgagggccagtccatcatgctgcacgtgcacatcaagcgccacgccatgaaggacctgcccgagtccgacgacgccgtcgcggagtggtgccgcgacaagttcgtcgagaaggacgccctgctggacaagcacaacgcggaggacacgttctccggccaggaggtgtgccactccggctcccgccagctgaagtccctgctggtcgtgatctcctgggtcgtggtgacgacgttcggcgccctgaagttcctgcagtggtcctcctggaagggcaaggcgttctccgccatcggcctgggcatcgtcaccctgctgatgcacgtgctgatcctgtcctcccaggccgagcgctccaaccccgccgaggtggcccaggccaagctgaagaccggcctgtccatctccaagaaggtgacggacaaggagaacTGAttaattaactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctctt cg pSZ2300 Transforming Construct gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactagaaccccacttgcactctgcaatggtatgggaaccacggggcagtcttgtgtgggtcgcgcctatcgcggtcggcgaagaccgggaag

aggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc CGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTC  

cttcgtgtccggcctgatcgtcaacctggtgcaggccgtctgcttcgtcctgatccgccccctgtccaagaacacgtaccgccgcatcaaccgcgtggtcgcggagctgctgtggctggagctggtgtggctgatcgactggtgggcgggcgtgaagatcaaggtcttcacggaccacgagacgttccacctgatgggcaaggagcacgccctggtcatctgcaaccacaagtccgacatcgactggctggtcggctgggtcctgggccagcgctccggctgcctgggctccaccctggcggtcatgaagaagtcctccaagttcctgcccgtcctgggctggtccatgtggttctccgagtacctgttcctggagcgctcctgggccaaggacgagatcacgctgaagtccggcctgaaccgcctgaaggactaccccctgcccttctggctggcgctgttcgtggagggcacgcgcttcacccgcgcgaagctgctggcggcgcagcagtacgccgcgtcctccggcctgcccgtgccccgcaacgtgctgatcccccgcacgaaggcttcgtgtcctccgtgtcccacatgcgctccttcgtgcccgcgatctacgacgtcaccgtggccatccccaagacgtcccccccccccacgctgatccgcatgttcaagggccagtcctccgtgctgcacgtgcacctgaagcgccacctgatgaaggacctgcccgagtccgacgacgccgtcgcgcagtggtgccgcgacatcttcgtggagaaggacgcgctgctggacaagcacaacgccgaggacaccttctccggccaggagctgcaggagaccggccgccccatcaagtccctgctggtcgtcatctcctgggccgtcctggaggtgttcggcgccgtcaagttcctgcagtggtcctccctgctgtcctcctggaagggcctggcgttctccggcatcggcctgggcgtgatcaccctgctgatgcacatcctgatcctgttctcccagtccgagcgctccacccccgccaaggtggcccccgcgaagcccaagaacgagggcgagtcctccaagaccgagatggagaaggagaagTGAttaattaactcgaggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttctta gctcttcg

To determine the impact of the CuPSR23 LPAAT2 and LPAAT3 genes onmid-chain fatty acid accumulation, the above constructs containing thecodon optimized CuPSR23 LPAAT2 or LPAAT3 genes driven by the UTEX 1453AMT3 promoter were transformed into strain B.

Primary transformants were clonally purified and grown under standardlipid production conditions at pH7.0 (all the strains require growth atpH 7.0 to allow for maximal expression of the CuPSR23 LPAAT2 or LPAAT3gene driven by the pH-regulated AMT3 promoter). The resulting profilesfrom a set of representative clones arising from these transformationsare shown in Table 34, below. D1520 represents clones of Strain B withCuPSR23 LPAAT2 and D1521 represents clones of Strain B with CuPSR23LPAAT3.

TABLE 34 Fatty acid profiles of Strain B and representative transgeniclines transformed with pSZ2299 and pSZ2300 DNA. Sample ID C10:0 C12:0C14:0 C16:0 C18:0 C18:1 C18:2 Strain B 4.83 28.54 15.64 12.64 1.3 27.997.75 D1520-A 8.59 35.09 16.55 11.96 1.69 19.49 5.59 D1520-B 8.13 33.9316.46 12.44 1.57 20.66 5.96 D1520-C 7.6 33.1 16.21 12.65 1.5 21.41 6.48D1520-D 7.35 32.54 16.03 12.79 1.67 22.16 6.41 D1520-E 7.28 32.21 16.212.99 1.73 22.39 6.28 D1521-A 6.14 31.5 15.98 12.96 1.96 22.52 8 D1521-B6.17 31.38 15.98 12.87 2.08 22.54 7.92 D1521-C 5.99 31.31 15.75 12.792.23 22.45 8.36 D1521-D 5.95 31.05 15.71 12.84 2.48 22.69 8.32 D1521-E5.91 30.58 15.85 13.22 1.97 23.55 7.84

The transgenic CuPSR23 LPAAT2 strains (D1520A-E) show a significantincrease in the accumulation of C10:0, C12:0, and C14:0 fatty acids witha concomitant decrease in C18:1 and C18:2. The transgenic CuPSR23 LPAAT3strains (D1521A-E) show a significant increase in the accumulation ofC10:0, C12:0, and C14:0 fatty acids with a concomitant decrease inC18:1. The expression of the CuPSR23 LPAAT in these transgenic linesappears to be directly responsible for the increased accumulation ofmid-chain fatty acids in general, and especially laurates. While thetransgenic lines show a shift from longer chain fatty acids (C16:0 andabove) to mid-chain fatty acids, the shift is targeted predominantly toC10:0 and C12:0 fatty acids with a slight effect on C14:0 fatty acids.The data presented also show that co-expression of the LPAAT2 and LPAAT3genes from Cuphea PSR23 and the FATB2 from C. wrightii (expressed in thestrain Strain B) have an additive effect on the accumulation of C12:0fatty acids.

Our results suggest that the LPAAT enzymes from Cuphea PSR23 are activein the algal strains derived from UTEX 1435. These results alsodemonstrate that the enzyme functions in conjunction with theheterologous FatB2 acyl-ACP thioesterase enzyme expressed in Strain B,which is derived from Cuphea wrightii.

Example 44 Alteration of Fatty Acid Levels in Strain UTEX1435 by theExpression of Cuphea PSR23 LPAATx in Combination with Cuphea WrightiiFatb2

Here we demonstrate the effect of expression of a1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) in a previouslydescribed P. moriformis (UTEX 1435) transgenic strain, Strain B. Asdescribed above, Strain B is a transgenic strain expressing the acyl ACPthioesterase (FATB2) from Cuphea wrightii, which accumulates C12:0 fattyacids between 40 to 49%. Further to Example 43, a third CuPSR23 LPAAT,LPAATx, was identified by analysis of a combination of CuPSR23 genomicsequences and transcriptomic sequences derived from seed RNAs.Expression of a mid-chain specific LPAAT should thus increase thepercentage of TAGs that have a capric acid (C10:0 fatty acid), lauricacid (C12:0 fatty acid), or myrisitc acid (C14:0 fatty acid) at the sn-2position, and should consequently elevate the overall levels of thesefatty acids. In Example 43, LPAAT2 and LPAAT3 were shown to increasecaprate, laurate, and myristate accumulation in strain B. LPAATx wasintroduced into strain B to determine its effect on fatty acid levels inthis strain. The LPAATx gene was codon optimized to reflect UTEX 1435codon usage. Transformations, cell culture, lipid production and fattyacid analysis were all carried out as previously described.

Decreased Caprate, Laurate, and Myristate Accumulation and IncreasedPalmitate and Stearate Accumulation in Strain Strain B by the Expressionof the Cuphea PSR231-acyl-sn-glycerol-3-phosphate acyltransferase(LPAATx) [pSZ2575]:

In this example, transgenic strains were generated via transformation ofstrain B with the construct pSZ2575 encoding CuPSR23 LPAATx. Thetransgenic strains were selected for resistance to the antibiotic G418.Construct pSZ2575 can be written aspLOOP5′::CrTUB2:NeoR:CvNR::PmAMT3:CuPSR23LPAATx:CvNR::pLOOP3′. Thesequence of the transforming DNA is provided below (pSZ2575). Therelevant restriction sites in the construct from 5′-3′, BspQ1, KpnI,XbaI, MfeI, BamHI, EcoRI, SpeI, XhoI, SacI, BspQ1, respectively, areindicated in lowercase, bold, and underlined. BspQ1 sites delimit the 5′and 3′ ends of the transforming DNA. Bold, lowercase sequences at the 5′and 3′ end of the construct represent genomic DNA from UTEX 1435 thattarget integration to the pLoop locus via homologous recombination.Proceeding in the 5′ to 3′ direction, the selection cassette has the C.reinhardtii β-tubulin promoter driving expression of the NeoR gene(conferring resistance to G418) and the Chlorella vulgaris NitrateReductase (NR) gene 3′ UTR. The promoter is indicated by lowercase,boxed text. The initiator ATG and terminator TGA for NeoR are indicatedby uppercase italics, while the coding region is indicated withlowercase italics. The 3′ UTR is indicated by lowercase underlined text.The spacer region between the two cassettes is indicated by upper casetext. The second cassette containing the codon optimized LPAATx gene(pSZ2575) from Cuphea PSR23 is driven by the Prototheca moriformisendogenous AMT3 promoter, and has the same Chlorella vulgaris NitrateReductase (NR) gene 3′ UTR. In this cassette, the AMT3 promoter isindicated by lowercase, boxed text. The initiator ATG and terminator TGAfor the CuPSR23 LPAATx genes are indicated in uppercase italics, whilethe coding region is indicated by lowercase italics. The 3′ UTR isindicated by lowercase underlined text. The final construct wassequenced to ensure correct reading frame and targeting sequences.

pSZ2575 Transforming Construct gctcttccgctaacggaggtctgtcaccaaatggaccccgtctattgcgggaaaccacggcgatggcacgtttcaaaacttgatgaaatacaatattcagtatgtcgcgggcggcgacggcggggagctgatgtcgcgctgggtattgcttaatcgccagcttcgcccccgtcttggcgcgaggcgtgaacaagccgaccgatgtgcacgagcaaatcctgacactagaagggctgactcgcccggcacggctgaattacacaggcttgcaaaaataccagaatttgcacgcaccgtattcgcggtattttgttggacagtgaatagcgatgcggcaatggcttgtggcgttagaaggtgcgacgaaggtggtgccaccactgtgccagccagtcctggcggctcccagggccccgatcaagagccaggacatccaaactacccacagcatcaacgccccggcctatactcgaaccccacttgcactctgcaatggtatgggaaccacggggcagtcttgtgtgggtcgcgcctatcgcggtcggcgaagaccgggaag

aggacggcctccacgccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcc CGCGTCTCGAACAGAGCGCGCAGAGGAACGCTGAAGGTCTCGCCTCTGTCGCACCTCAGCGCGGCATACACCACAATAACCACCTGACGAATGCGCTTGGTTCTTCGTCCATTAGCGAAGCGTCCGGTTCACACACGTGCCACGTTGGCGAGGTGGCAGGTGACAATGATCGGTGGAGCTGATGGTC

ccagctgtactacaagaagaagaagcacgccatcctgcagacccagaccccctaccgctaccgcgtgtcccccacctgcttcgcccccccccgcctgcgcaagcagcacccctaccccctgcccgtgctgtgctaccccaagctgctgcacttctcccagccccgctaccccctggtgcgctcccacctggccgaggccggcgtggcctaccgccccggctacgagctgctgggcaagatccgcggcgtgtgcttctacgccgtgaccgccgccgtggccctgctgctgttccagtgcatgctgctgctgcaccccttcgtgctgctgttcgaccccttcccccgcaaggcccaccacaccatcgccaagctgtggtccatctgctccgtgtccctgttctacaagatccacatcaagggcctggagaacctgccccccccccactcccccgccgtgtacgtgtccaaccaccagtccttcctggacatctacaccctgctgaccctgggccgcaccttcaagttcatctccaagaccgagatcttcctgtaccccatcatcggctgggccatgtacatgctgggcaccatccccctgaagcgcctggactcccgctcccagctggacaccctgaagcgctgcatggacctgatcaagaagggcgcctccgtgttcttcttccccgagggcacccgctccaaggacggcaagctgggcgccttcaagaagggcgccttctccatcgccgccaagtccaaggtgcccgtggtgcccatcaccctgatcggcaccggcaagatcatgccccccggctccgagctgaccgtgaaccccggcaccgtgcaggtgatcatccacaagcccatcgagggctccgacgccgaggccatgtgcaacgaggcccgcgccaccatctcccactccctggacgacTGAttaattaactcgaggcagcagcagctcggatagtgatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagctt gagctcagcggcgacggtcctgctaccgtacgacgttgggcacgcccatgaaagtttgtataccgagcttgttgagcgaactgcaagcgcggctcaaggatacttgaaactcctggattgatatcggtccaataatggatggaaaatccgaacctcgtgcaagaactgagcaaacctcgttacatggatgcacagtcgccagtccaatgaacattgaagtgagcgaactgttcgcttcggtggcagtactactcaaagaatgagctgctgttaaaaatgcactctcgttctctcaagtgagtggcagatgagtgctcacgccttgcacttcgctgcccgtgtcatgccctgcgccccaaaatttgaaaaaagggatgagattattgggcaatggacgacgtcgtcgctccgggagtcaggaccggcggaaaataagaggcaacacactccgcttcttagctcttcg

To determine the impact of the CuPSR23 LPAATx gene on fatty acidaccumulation, the above construct containing the codon optimized CuPSR23LPAATx gene driven by the UTEX 1453 AMT3 promoter was transformed intostrain B.

Primary transformants were clonally purified and grown under lownitrogen conditions at pH7.0; the strains require growth at pH 7.0 toallow for maximal expression of the CuPSR23 LPAATx and CwFATB2 genesdriven by the pH-regulated AMT3 promoter. The resulting profiles from aset of representative clones arising from these transformations areshown in Table 35, below. D1542 represents clones of Strain B withCuPSR23 LPAATx.

TABLE 35 Fatty acid profiles of Strain B and representative transgeniclines transformed with pSZ2575. Sample ID C10:0 C12:0 C14:0 C16:0 C18:0C18:1 C18:2 Strain 4.77 28.63 15.48 12.65 1.28 28.20 7.57 B D1542- 1.1913.25 10.48 21.34 4.49 32.07 14.78 A D1542- 1.15 14.01 10.62 20.61 3.9932.12 15.24 B D1542- 1.21 13.69 10.83 20.40 3.59 33.54 15.05 C D1542-1.56 16.83 11.51 18.44 2.94 33.97 12.74 D D1542- 2.15 18.58 11.94 18.223.17 32.63 11.62 E

The transgenic CuPSR23 LPAATx strains (D1542A-E) show a significantdecrease in the accumulation of C10:0, C12:0, and C14:0 fatty acidsrelative to the parent, Strain B, with a concomitant increase in C16:0,C18:0, C18:1 and C18:2. The expression of the CuPSR23 LPAATx gene inthese transgenic lines appears to be directly responsible for thedecreased accumulation of mid-chain fatty acids (C10-C14) and theincreased accumulation of C16:0 and C18 fatty acids, with the mostpronounced increase observed in palmitates (C16:0). The data presentedalso show that despite the expression of the midchain specific FATB2from C. wrightii (present in Strain B), the expression of CuPSR23 LPAATxappears to favor incorporation of longer chain fatty acids into TAGs.

Our results suggest that the LPAATx enzyme from Cuphea PSR23 is activein the algal strains derived from UTEX 1435. Contrary to Cuphea PSR23LPAAT2 and LPAAT3, which increase mid-chain fatty acid levels, CuPSR23LPAATx leads to increased C16:0 and C18:0 levels. These resultsdemonstrate that the different LPAATs derived from CuPSR23 (LPAAT2,LPAAT3, and LPAATx) exhibit different fatty acid specificities in StrainB as judged by their effects on overall fatty acid levels.

Example 45 Reduction in Chain Length of Fatty Acid Profile as a Resultof Overexpressing an Endogenous Microalgal FATA Acyl-ACP Thioesterase

Here, we demonstrate that over expression of the Prototheca moriformisendogenous thioesterases FATA1 in UTEX1435 results in a clear diminutionof cell triglyceride C18:0 and C18:1 acyl chains with an increase inC16:0, C14:0.

Constructs Used for the Over Expression of the P. moriformis FATA1 Gene(pSZ2422, pSZ2421):

To over express the PmFATA1 in P. moriformis STRAIN J, a codon optimizedPmFATA1 gene was been transformed into STRAIN J. The Saccharomycescerevisiae invertase gene was utilized as the selectable marker toconfer the ability of growing on sucrose media. The construct pSZ2422that have been expressed in STRAIN J can be written as:6SA::CrTUB2-ScSUC2-CvNR3′:PmAMT3-Pm FATA1 (opt)-CvNR3′::6SB, and theconstruct pSZ2421 can be written as6SA::CrTUB2-ScSUC2-CvNR3′:PmAMT3-S106SAD TP-Pm FATA1 (opt)-CvNR3′::6SB.

The sequence of the transforming DNA is provided below. Relevantrestriction sites in the construct pSZ2422 are indicated in lowercase,bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, Mfe I, BamH I,EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively. BspQI sitesdelimit the 5′ and 3′ ends of the transforming DNA. Bold, lowercasesequences represent genomic DNA from STRAIN J that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof STRAIN J to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of P. moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the PmFATA1 are indicated byuppercase, bold italics, while the remainder of the gene is indicated bybold italics. The C. vulgaris nitrate reductase 3′ UTR is againindicated by lowercase underlined text followed by the STRAIN J 6Sgenomic region indicated by bold, lowercase text.

Relevant restriction sites in the construct pSZ2421 are the same aspSZ2422. In pSZ2421, the PmFATA1 is fused to the Chlorellaprotothecoides S106 stearoyl-ACP desaturase transit peptide and thetransit peptide is located between initiator ATG of PmFATA1 and the AscI site.

Nucleotide sequence of transforming DNA contained in pSZ2422 gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgcccagccgctgggggttggcggatgcacgctcaggta

ccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctcggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaag TGAcaattggcagcagcagctcggatagtatcgacacactctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggc

ctctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgctttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatccasaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttgttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

To determine the impact on fatty acid profiles when the endogenous FATA1gene have been over expressed in STRAIN J, both the P. moriformis FATA1with native transit peptide and PmFATA1 fused to a Chlorellaprotothecoides SAD transit peptide were driven by the amt03 promoter andthe resulting plasmids were transformed independently into STRAIN J.

Primary transformants were clonally purified and grown underlow-nitrogen lipid production conditions at pH7.0 (all the plasmidsrequire growth at pH 7.0 to allow for maximal PmFATA1 gene expressionwhen driven by the pH regulated amt03 promoter). The resulting profilesfrom representative clones arising from transformations with pSZ2422 andpSZ2421 into STRAIN J are shown in the tables below.

In Table 36, below, the impact of over expressing native PmFATA1 is aclear diminution of C18:1 chain lengths with an increase in C16:0,C14:0, and possibly in C18:0. Considering the protein localization ofprocessing, we also tried the PmFATA1 fused to a Chlorellaprotothecoides stearoyl-ACP desaturase transit peptide. Similar to theresults we observed in the amt03-native PmFATA1 construct, the C16:0 andC14:0 levels are significantly higher than the parental strain STRAIN J.

TABLE 36 Fatty acid profiles in Strain J and derivative transgenic linestransformed with pSZ2422 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH7; Strain J; T374; 7.69 55.00 4.92 24.94 5.19 D1377-7 96well pH 7;Strain J; T374; 6.39 54.11 5.85 25.91 5.76 D1377-13 96well pH 7; StrainJ; T374; 6.57 53.55 4.68 27.18 5.74 D1377-14 96well pH 7; Strain J;T374; 5.29 49.93 4.24 30.76 7.27 D1377-16 96well pH 7; Strain J; T374;4.76 49.10 4.75 32.36 6.77 D1377-9 96well pH 7; Strain J; T374; 4.2846.06 5.14 35.87 6.69 D1377-19 96well Ctrl-pH7; Strain J 1.42 27.63 3.3157.20 8.00

TABLE 37 Fatty acid profiles in STRAIN J and derivative transgenic linestransformed with pSZ2421 DNA. Sample ID C14:0 C16:0 C18:0 C18:1 C18:2 pH7; STRAIN J; T374; 6.76 57.06 4.12 23.66 6.07 D1376-21 96well pH 7;STRAIN J; T374; 6.56 54.62 5.44 25.69 5.64 D1376-22 96well pH 7; STRAINJ; T374; 4.54 48.38 4.27 33.23 7.24 D1376-23 96well pH 7; STRAIN J;T374; 4.48 47.66 4.60 34.28 6.91 D1376-19 96well pH 7; STRAIN J; T374;4.53 47.30 4.67 34.51 6.80 D1376-20 96well pH 7; STRAIN J; T374; 3.5642.70 4.03 39.85 7.52 D1376-17 96well Ctrl-pH7; STRAIN J 1.42 27.63 3.3157.20 8.00

Thus, we conclude that percent myristic and lauric acid levels in thefatty acid profile of a microalgal cell can be increased byoverexpression of a C18-preferring acyl-ACP thioesterase.

Example 46 Natural Oils Suitable for Use as Roll-in Shortenings

The nutritional and functional properties of edible fats have beentraditionally associated with specific chemical compositions andcrystallization conditions. Switching from one oil source to another isusually a difficult task since both the melting behavior and structureof the fat changes dramatically, leading to adverse changes infunctionality. In recent history, we can recall the painful period whenpartially hydrogenated fats were replaced with palm oil and palm oilfractions. We examined how the yield stress, elastic modulus,polymorphism, microstructure and melting profile of two fats with vastlydifferent chemical compositions can be matched. Oil A was produced fromPrototheca moriformis cells expressing an exogenous invertase and anUlmus americana acyl-ACP thioesterase with a Chlorella protothecoidesplastid targeting sequence. Oil B was produced from Protothecamoriformis cells expressing an exogenous invertase and a Cupheahookeriana acyl-ACP thioesterase. Oil A contained greater than 62% (w/w)medium chain fatty acids, or MCT (C8:0-C14:0), 23% (C16:0+C18:0) and 9%C18:1, while Oil B contained less than 2% C8:0-C14:0, 54% (C16:0+C18:0)and 29% C18:1. Oil A was thus a medium chain triglyceride rich fat,while Oil B resembled palm oil. Both oils had a solid fat content of˜45% at 20° C., and very similar SFC versus temperature profiles. DSC(dynamic scanning calorimetry) melting profiles showed two major peakscentered around ˜12-13° C. and ˜28-35° C. Both fats were in thebeta-prime polymorphic form (as determined by X-ray diffraction) anddisplayed asymmetric, elongated crystallite morphology withcharacteristic features. The yield stresses and storage moduli (G′) ofOil A and Oil B were 520-550 Pa, and 7×10⁶ Pa-1.8×10⁷ Pa, respectively.A yield stress in this region suggests a satisfactory plasticity, whichcombined with a high storage modulus makes for an ideal roll-inshortening. Thus, it is possible to alter the chemical composition of afood oil while retaining its lamination functionality.

Other suitable enzymes for use with the cells and methods of any of theabove embodiments of the invention include those that have at least 70%amino acid identity with one of the proteins listed in the descriptionabove and that exhibit the corresponding desired enzymatic activity. Inadditional embodiments, the enzymatic activity is present in a sequencethat has at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 99% identity withone of the above described nucleic acid sequences, all of which arehereby incorporated by reference as if fully set forth.

Example 47 Fractionation to Remove Trisaturates from a TailoredMicrobial Oil that is a Cocoa Butter Mimetic

A refined bleached and deodorized oil was obtained from Strain K4 (seeExample 35). The oil was heated to 70° C. and cooled at 0.5° C. per minto 36° C. and held at 36° C. for 1 hour. An approximately 2.5 ml samplewas then centrifuged at 36° C. for 1 hour at 4300. A liquid supernatantwas recovered and analysed using lipase and mass spectrometry. Thesample was found to be depleted in tristearin (SSS), SSP, and PPS. Thetriacylglycerols of the sample were found to be very similar to that ofcocoa butter and the liquid supernatant was even closer to that of cocoabutter in terms of low amounts of trisaturates.

TABLE 38 TAG profile of oil from the K4 strain before and afterfractionation as compared to cocoa butter. fractionation upper layer TAGK4 oil (liquid) cocoa butter OOL (+?) 0.12 0.12 0.00 POL 0.23 0.31 0.33PLP 2.41 3.38 1.58 MOP 0.93 1.25 0.00 PPM (+ 0.42 0.29 0.00 MMS) OOO0.23 0.34 0.00 SOL 0.36 0.47 0.32 OOP 0.95 1.42 2.44 PLS 5.66 7.90 2.90POP (+ 11.80 15.20 17.93 MSO) PPP + MPS 2.22 1.07 0.36 OOS 1.19 1.683.02 SLS (+ PLA) 3.96 5.11 1.77 POS 27.22 32.80 40.25 PPS (+ SSM) 6.471.52 0.49 MaOO 0.00 0.00 0.36 SLA 0.31 0.34 0.00 SOS (+ POA) 17.84 22.5024.93 SSP (+ PPA) 9.24 0.96 0.63 SOA (+ POB) 1.39 1.68 1.51 SSS (+ PSA)5.25 0.23 0.33 SOB + LgOP 0.38 0.44 0.27 SSA 0.41 0.00 0.00 SOLg 0.410.00 0.00 PSLg + ASB 0.26 0.00 0.00 SOHx 0.12 0.51 0.00 SSLg 0.21 0.140.15 SUM area % 100.00 99.67 99.57

Example 48 Production of High-Stearate Triglyceride Oil in an OleaginousCell by Overexpression of KASII, Knockout of One Sad Allele andRepression of a Second Sad Allele

The oleaginous, non-photosynthetic alga, Protetheca moriformis, storescopious amounts of triacylglyceride oil under conditions where thenutritional carbon supply is in excess, but cell division is inhibiteddue to limitation of other essential nutrients. Bulk biosynthesis offatty acids with carbon chain lengths up to C18 occurs in the plastids;fatty acids are then exported to the endoplasmic reticulum whereelongation past C18 and incorporation into triacylglycerides (TAGs) isbelieved to occur. Lipids are stored in large cytoplasmic organellescalled lipid bodies until environmental conditions change to favorgrowth, whereupon they are rapidly mobilized to provide energy andcarbon molecules for anabolic metabolism. Wild-type P. moriformisstorage lipid is mainly comprised of ˜60% oleic (C18:1), ˜25-30%palmitic (C16:0), and ˜5-8% linoleic (C18:2) acids, with minor amountsof stearic (C18:0), myristic (C14:0), α-linolenic (C18:3α), andpalmitoleic (C16:1) acids. This fatty acid profile results from therelative activities and substrate affinities of the enzymes of theendogeneous fatty acid biosynthetic pathway. P. moriformis is amenableto manipulation of fatty acid and lipid biosynthesis using moleculargenetic tools, enabling the production of oils with fatty acid profilesthat are very different to the wild-type composition. Herein we describestrains where we have modified the expression of stearoyl-ACP desaturase(SAD) and β-ketoacyl-ACP synthase II (KASII) genes in order to generatestrains with up to 57% stearate and as little as 7% palmitate. Weidentify additional strains with up to 55% stearate and as low as 2.4%linoleate when we perform similar modifications in conjunction withdown-regulating the expression of the FATA thioesterase and the FAD2fatty acid desaturase genes.

Soluble SADs are plastid-localized, di-iron enzymes which catalyze thedesaturation of acyl carrier protein (ACP)-bound stearate to oleate(C18:1 cis-Δ⁹). Previously, we have established that hairpin constructstargeting the SAD1 or SAD2 transcripts activate the cellular RNAinterference (RNAi) machinery, down-regulating SAD activity andresulting in elevated levels of C18:0 in the storage lipid. SAD activityis also reduced in strains where we disrupt one of the two alleles ofSAD2, encoding the major SADs that are expressed during storage lipidbiosynthesis. The Fatty Acid Desaturase 2 (FAD2) gene encodes anendoplasmic reticulum membrane-associated desaturase that convertsoleate to linoleate (C18:2 cis-Δ⁹, cis-Δ¹²). Hairpin RNAi constructstargeting FAD2 reduce linoleate levels to 1-2%. KASII is a fatty acidsynthase which specifically catalyzes the condensation of malonyl-ACPwith palmitoyl (C16:0)-ACP to form β-keto-stearoyl-ACP. We have shownthat overexpression of KASII in P. moriformis causes C16:0 levels todecrease with a concommitent increase in C18:1 abundance. In theexamples below we demonstrate that by down-regulating SAD geneexpression using RNAi, disrupting an allele of the SAD2 gene, andoverexpressing the KASII fatty acid synthase, we generate strainscapable of accumulating stearate in excess of 50% of the total fattyacids, and with SOS as the major TAG species. SOS levels increase up to47% in strains which combine SAD2 and FAD2 down-regulation with KASIIoverexpression.

Constructs Used for SAD2 Knockout/RNAi in S1920:

A DNA construct, pSZ2282, was made to simultaneously disrupt the SAD2-1allele and express a SAD2 hairpin construct in S1920. A version of theSaccharomyces cerevisiae SUC2 gene, encoding sucrose invertase, whichwas codon-optimized for expression in P. moriformis, was utilized as aselectable marker for transformation. The sequence of the transformingDNA is provided immediately below. Relevant restriction sites areindicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, AscI,MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI,respectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Underlined sequences at the 5′ and 3′ flanks of the constructrepresent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the Chlamydomonasreinhardtii TUB2 promoter driving the expression of the Saccharomycescerevisiae SUC2 gene (encoding sucrose hydrolyzing activity, therebypermitting the strain to grow on sucrose) is indicated by lowercase,boxed text. The initiator ATG and terminator TGA for SUC2 are indicatedby uppercase italics, while the coding region is indicated withlowercase italics. The 3′ UTR of the Chlorella vulgaris nitratereductase (NR) gene is indicated by small capitals, followed by a spacerregion indicated by lowercase text. A second C. reinhardtii TUB2promoter sequence, indicated by lowercase boxed text, drives expressionof the SAD2 hairpin C sequence. The sense and antisense strands areindicated with uppercase, bold italics, and are separated by the P.moriformis Δ¹²-fatty acid desaturase (FAD2) intron and the first 10bases of the FAD2 second exon (uppercase italics). A second C. vulgarisNR 3′ UTR is indicated by small capitals.

Nucleotide sequence of the transforming DNA from pSZ2282: gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgca

ccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgcccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagcggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtc

GTGTTTGAGGGTTTTGGTTGCCCGTATCGAGGTCCTGGTGGCGCGCATGGGGGAGAAGGCGCCT

ACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCTGgagctccagccacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctagccaacatgatgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaactcttcgccgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaggtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagggagcgacgagtgtgcgtgcaggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactcct gaagagc

Identification and Analysis of SAD2 Knockout/Knockdown Strains:

Construct D1283, derived from pSZ2282, was transformed into S1920 asdescribed previously. Primary transformants were clonally purified andgrown under standard lipid production conditions at pH 5. The resultingfatty acid profiles from representative clones arising fromtransformation with pSZ2282 into S1920 are summarized in Table 39,below. D1283 transformants accumulated up to ˜42% C18:0 at the expenseof C18:1, indicating that SAD activity was significantly reduced inthese strains.

TABLE 39 Fatty acid profiles of D1283 [pSZ2282] primary transformants,compared to the wild-type parental strain, S1920. Strain S1920 D1283-4D1283-7 D1283-19 D1283-27 D1283-40 D1283-24 Fatty C12:0 0.04 0.05 0.060.07 0.06 0.04 0.05 Acid C14:0 1.31 0.92 1.07 1.01 1.08 1.03 0.96 AreaC16:0 26.68 28.23 29.21 27.24 27.67 27.02 27.07 % C16:1 0.78 0.05 0.060.08 0.33 0.14 0.12 C17:0 0.11 0.12 0.15 0.10 0.10 0.12 0.13 C18:0 3.1541.98 40.94 34.20 26.26 23.18 22.82 C18:1 59.30 19.37 18.17 26.87 34.7738.74 39.38 C18:2 7.47 6.22 7.43 7.42 7.31 7.25 7.38 C18:3α 0.57 0.931.03 0.75 0.71 0.72 0.51 C20:0 0.32 1.81 1.67 1.75 1.35 1.36 1.23 C20:10.00 0.10 0.00 0.12 0.00 0.12 0.11 C22:0 0.05 0.17 0.13 0.20 0.16 0.160.15 C24:0 0.00 0.00 0.00 0.10 0.00 0.00 0.00 sum C18 70.49 68.5 67.5769.24 69.05 69.89 70.09 saturates 31.66 73.28 73.23 64.67 56.68 52.9152.41 unsaturates 68.12 26.67 26.69 35.24 43.12 46.97 47.50

In Table 39, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text.

The fatty acid profiles of transformants D1283-4 and -7 were determinedto be stable after more than 30 generations of growth in the absence ofselection (growth on sucrose). The performance of selected strains inshake flask assays was then evaluated, and the fatty acid profiles andlipid titers are presented in Table 40, below. S4495 had the highestlevel of C18:0 (˜44%) and the best lipid titer (˜26%) relative to theS1920 parent, and so was selected for further fermentation development.

TABLE 40 Fatty acid profiles and lipid titers of SAD2knockout/knock-down strains derived from D1283 primary transformants,compared to the wild-type parental strain, S1920. Primary T342; D1283-4T342; D1283-7 Strain S1920 S4490 S4491 S4492 S4493 S4494 S4495 FattyC14:0 1.59 1.61 1.58 1.55 1.81 1.84 1.34 Acid C16:0 30.47 29.41 28.5829.24 28.77 29.09 28.47 Area C16:1 0.82 0.05 0.07 0.05 0.07 0.05 0.06 %C17:0 0.10 0.30 0.29 0.28 0.46 0.37 0.19 C18:0 3.58 42.85 41.86 43.3839.99 41.41 44.42 C18:1 56.96 13.52 15.55 13.49 13.57 12.98 15.64 C18:25.50 8.01 7.85 7.65 10.37 9.47 5.72 C18:3α 0.37 0.78 0.73 0.82 0.95 0.910.64 C20:0 0.22 2.06 2.11 2.11 1.98 1.98 2.32 C22:0 0.05 0.32 0.34 0.330.33 0.32 0.35 C24:0 0.03 0.43 0.42 0.44 0.49 0.49 0.37 lipid titer (%parent) 100 12.3 12.6 13.6 6.2 8.2 25.9

In Table 40, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text.

We optimized the performance of S4495 in 7-L fermentations, and foundthat we could match the ˜44% C18:0 level obtained in shake flasks, withlipid productivities that were ˜45% of the wild-type parent. The fattyacid profiles and lipid titers of representative S4495 fermentations aresummarized in Table 41, below. Fermentation of S4495 under optimalconditions yielded nearly 44% C18:0, which was similar to the stearatelevel that accumulated in shake flask assays. S4495 produced high C18:0levels at both flask and 7-L scale and had acceptable lipid productivityin 7-L fermentations; consequently this strain was selected as a basestrain for additional modifications aimed at increasing C18:0accumulation.

TABLE 41 Fatty acid profiles and lipid titers of S4495, compared to acontrol transgenic strain S2074. Strain S2074 S4495 S4495 S4495Fermentation 110088F14 120489F5 120531F8 120580F1 Fatty C14:0 14.7 1.181.15 1.27 Acid C16:0 25.66 28.68 28.38 28.35 Area % C16:1 0.71 0.11 0.090.06 C18:0 3.16 41.63 42.40 43.67 C18:1 62.24 20.78 19.38 17.63 C18:25.90 5.06 5.38 5.58 C18:3α 0.16 0.24 0.25 0.25 C20:0 0.24 1.36 1.99 2.11C22:0 0.05 0.19 0.28 0.31 C24:0 0.05 0.34 0.29 0.31 sum C18 71.46 67.7167.41 67.13 saturates 30.63 73.38 74.49 76.02 unsaturates 69.01 26.1925.10 23.52 total lipid (g/L) 930 383 539 475

In Table 41, Stearate (C18:0) levels greater than the control arehighlighted with bold text. S2074 contains S. cerevisiae SUC2, encodingsucrose invertase, integrated at the 6S locus, and has a fatty acidprofile that is indistinguishable from the S1920 wild-type parent.

Constructs Used for KASII Overexpression in S4495:

DNA construct pSZ2734 was made to overexpress a codon-optimized P.moriformis KASII gene in S4495. The neoR gene from transposon Tn5,conferring resistance to aminoglycoside antibiotics, was used as aselectable marker for transformation. The sequence of the transformingDNA is provided immediately below. Relevant restriction sites areindicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, XbaI,MfeI, BamHI, AvrII, EcoRV, SpeI, AscI, ClaI, BglII, AflII, HinDIII andSacI, respectively. BspQI sites delimit the 5′ and 3′ ends of thetransforming DNA. Underlined sequences at the 5′ and 3′ flanks of theconstruct represent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at the6S locus. Proceeding in the 5′ to 3′ direction, the C. reinhardtii TUB2promoter driving the expression of neoR (encoding aminoglycosidephosphotransferase activity, thereby permitting the strain to grow onG418) is indicated by lowercase, boxed text. The initiator ATG andterminator TGA for neoR are indicated by uppercase italics, while thecoding region is indicated with lowercase italics. The 3′ UTR of the C.vulgaris NR gene is indicated by small capitals, followed by a spacerregion indicated by lowercase text. The P. moriformis SAD2-2 promotersequence, indicated by boxed text, drives expression of thecodon-optimized P. moriformis KASII gene. The region encoding the KASIIplastid targeting sequence is indicated by uppercase italics. Thesequence that encodes the mature P. moriformis KASII polypeptide isindicated with bold, underlined, uppercase italics, while a 3×FLAGepitope encoding sequence is in bold italics. A second C. vulgaris NR 3′UTR is indicated by small capitals.

Nucleotide sequence of the the transforming DNA from pSZ2734: gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggtgtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagag

agggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgtt

CACCAGCGCCCCCCCACCGAGGGCCACTGCTTCGGCGCCCGCCTGCCCACCGCCTCCCGCCGCGC

gatagatctcttaagGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagcttaattaagagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

Overexpression of KASII in Strain X:

Construct D1643 derived from pSZ2734 was transformed into S4495 asdescribed previously. Primary transformants were clonally purified andgrown under standard lipid production conditions at pH 5. The resultingfatty acid profiles from representative clones arising fromtransformation of S4495 with D1643 are summarized in Table 42, below.Overexpression of KASII in the SAD2 knockout/knock-down S4495 backgroundresulted in multiple strains accumulating over 50% C18:0 and withsubstantially reduced levels of C16:0. We also observed that KASIIover-expressing lines had lower overall ratios of saturated tounsaturated fatty acids compared to S4495.

TABLE 42 Fatty acid profiles of D1653 [pSZ27341] primary transformants,compared to the S4495 base strain and the wild-type parental strain,S1920. D1653- D1653- D1653- D1653- D1653- D1653- D1653- D1653- D1653-D1653- D1653- D1653- Strain S1920 S4495 89 10A 2B 5B 7A 75 90 9B 72 6B82 66 Fatty C12:0 0.04 0.06 0.27 0.13 0.20 0.19 0.24 0.13 0.12 0.27 0.160.18 0.25 0.22 Acid C14:0 1.44 1.06 1.55 1.65 1.79 1.67 1.70 1.53 1.501.74 1.57 1.64 1.48 1.58 Area C16:0 29.23 29.83 8.16 11.45 10.68 10.119.27 11.14 11.08 9.40 9.78 9.95 8.12 8.65 % C16:1 0.88 0.10 0.04 0.000.00 0.00 0.00 0.04 0.04 0.00 0.04 0.00 0.05 0.06 C18:0 2.97 40.17 54.2553.87 53.61 53.46 53.32 53.32 53.15 52.43 52.20 51.23 50.52 50.02 C18:158.07 20.15 23.52 22.12 22.20 23.48 24.02 22.73 23.45 23.94 25.21 26.0728.00 28.29 C18:2 6.25 5.25 6.75 6.05 6.42 6.25 6.56 6.19 5.96 6.88 6.286.31 6.59 6.31 C18:3α 0.50 0.68 0.79 0.88 0.78 0.79 0.79 0.85 0.82 0.860.78 0.78 0.78 0.83 C20:0 0.22 1.88 3.21 2.81 3.01 2.91 3.02 2.86 2.773.21 2.74 2.80 2.87 2.80 C20:1 0.02 0.07 0.19 0.21 0.34 0.27 0.28 0.120.11 0.41 0.14 0.30 0.28 0.26 C22:0 0.05 0.26 0.41 0.34 0.40 0.37 0.370.36 0.35 0.42 0.36 0.37 0.36 0.37 C24:0 0.04 0.27 0.49 0.38 0.42 0.410.45 0.38 0.36 0.46 0.39 0.37 0.41 0.41 sum C18 67.78 66.24 85.31 82.9283.01 83.98 84.69 83.09 83.38 84.11 84.47 84.39 85.89 85.45 saturates33.97 73.52 68.34 70.63 70.11 69.12 68.37 69.72 69.33 67.93 67.20

unsaturates 65.71 26.23 31.29 29.26 29.74 30.79 31.65 29.93 30.38 32.0932.45

In Table 42, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text. Palmitate (C16:0) levels lower thanS4495 or S1920 are highlighted with bold. For three strains the ratio ofsaturated to unsaturated fatty acids is ≦2:1; these are highlighted withbold, italicized text.

Stable lines were isolated from the primary transformants shown in Table42. The fatty acid profiles and lipid titers of shake flask cultures arepresented in Table 43, below. The strains accumulated up to 55% C18:0,with as low as 7% C16:0, with comparable lipid titers to the S4495parent. The saturates:unsaturates ratios were substantially reducedcompared to S4495. Strains S5665 and S5675 were selected for evaluationin 3-L high-density fermentations.

TABLE 43 Shake flask assays of strains derived from D1653, expressingKASII, driven by the PmSAD2-2 promoter, targeted to the 6S locus.Primary D1653-6B D1653-9B D1653-10A D1653-72 D1653-89 Strain S1920 S4495S5664 S5665 S5669 S5670 S5671 S5672 S5673 S5674 S5675 S5677 Fatty C10:00.02 0.04 0.08 0.09 0.12 0.06 0.06 0.08 0.09 0.12 0.12 0.12 Acid C12:00.04 0.09 0.28 0.29 0.35 0.20 0.20 0.23 0.26 0.32 0.32 0.33 Area C14:01.42 1.12 1.81 1.66 1.73 1.75 1.72 1.50 1.61 1.38 1.43 1.38 % C16:025.59 28.56 9.39 8.61 8.44 9.98 10.11 8.26 8.95 6.81 7.21 6.63 C16:11.03 0.10 0.06 0.05 0.06 0.06 0.06 0.04 0.04 0.03 0.03 0.03 C18:0 2.6040.13 47.60 52.47 55.12 50.25 49.73 54.56 54.01 52.96 53.68 52.12 C18:162.08 20.74 27.78 23.93 21.31 25.37 25.70 22.86 22.87 24.37 23.99 25.17C18.2 6.16 5.83 7.98 7.52 7.72 7.55 7.64 7.20 7.24 8.11 7.83 8.04 C18.3α0.40 0.89 1.21 1.22 1.49 1.17 1.07 1.20 1.29 1.28 1.24 1.31 C20:0 0.181.82 2.62 2.93 2.75 2.65 2.66 2.97 2.72 3.43 3.10 3.59 C20:1 0.04 0.130.37 0.36 0.39 0.34 0.34 0.35 0.34 0.48 0.41 0.47 C20:1 0.07 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 0.15 0.08 0.11 0.090.11 0.10 0.10 0.09 0.10 0.12 0.10 0.12 C22.0 0.02 0.20 0.28 0.30 0.240.29 0.28 0.30 0.27 0.32 0.29 0.35 C24:0 0.00 0.03 0.16 0.29 0.00 0.030.15 0.16 0.02 0.05 0.04 0.07 sum C18 71.23 67.58 84.57 85.13 85.6384.34 84.13 85.81 85.40 86.71 86.73 86.63 saturates 29.86 71.97

67.74

68.05 67.90

unsaturates 69.91 27.76

31.07

31.73 31.87

% parent 100.0 56.2 43.4 48.8 27.5 44.1 47.4 59.0 47.6 43.2 48.4 44.4lipid titer

In Table 43, S4495 is the parent strain; S1920 is the wild-type basestrain. Stearate (C18:0) levels at least two-fold higher than in thewild-type strain are highlighted in bold. Palmitate levels that are lessthan in S1920 and S4495 are highlighted bold. Bold italics indicate thatthe saturates:unsaturates ratio is ≦2:1.

The fatty acid profiles and performance metrics of strains S5665 andS5675 are detailed in Table 44, below. The fatty acid profile of theparent strain S4495, grown under the same fermentation conditions, ispresented for comparison. The strains that over-express KASII accumulateabout 11% more C18:0 than the S4495 parent. C16:0 is reduced to 7-9%,and levels of unsaturated fatty acids increase by 4-5%. The lipid titersof S5665 and S5675 were comparable to S4495, indicating that KASIIover-expression did not have deleterious effects on lipid production.

TABLE 44 End point fatty acid profiles of biomass from S4495, S5665 andS5775 fermentations. Strain S4495 S5665 S5675 Fermentation 120580F1130097F3 130098F4 pH 5 5 5 temp (° C.) 32 32 32 [N] (mM) 300 300 300 N/P1.4 1.4 1.4 DO % 30 30 30 sugar 570 570 570 Fe (μM) 557.5 557.5 557.5C14:0 1.27 1.50 1.35 C16:0 28.35 8.88 7.33 C16:1 0.06 0.02 0.03 C18:043.67 56.88 57.24 C18:1 17.63 21.57 21.66 C18:2 5.58 60.6 6.94 C18:3α0.25 0.29 0.22 C20:0 2.11 3.28 3.46 C22:0 0.31 0.40 0.40 C24:0 0.31 0.370.40 sum C18 67.13 84.80 86.06 saturates 76.02 71.31 70.18 unsaturates23.52 27.94 28.85 total lipid (g/L) 475 529 418

The fermentations were cultured for 6 days using a fed-batch process.The S4495 fatty acid profile from fermentation 120580F1 was presented inTable 41, and is shown again in Table 44 for comparison with S5665 andS5675. All fermentations were carried out at 32° C., pH 5, with anitrogen/phosphorus (N/P) ratio of 1.4, 30% dissolved oxygen (DO), 300mM nitrogen [N], and 557.5 μM iron. The sugar source was 70% sucrose(S70). Stearate (C18:0) levels higher than in the wild-type strain areindicated with bold. Palmitate (C16:0) levels that are less than in thewild-type are highlighted bold.

Lab scale oils were prepared from biomass derived from the shake flasksand fermentations described above. The TAG compositions of these oilswere determined by LC/MS. SOS is the major TAG species in both S5665 andS5675, ranging from 33-35% in the biomass from shake flasks, andreaching 37% in the high-density fermentation biomass. The majorpalmitate-containing TAGs are substantially reduced, and trisaturatelevels are less than half of those observed in S4495 oils. These resultsdemonstrate that KASII over-expression in a high-stearate backgroundsignificantly improves SOS accumulation, and reduces the accumulation oftrisaturated TAGs.

Constructs Used for FATA-1 Disruption, KASII Over-Expression and FAD2RNAi in S1920:

A DNA construct, pSZ2419, was made to simultaneously disrupt the FATA-1allele, over-express P. moriformis KASII and express a FAD2 hairpinconstruct in S1920. A version of the S. cerevisiae SUC2 gene, encodingsucrose invertase, which was codon-optimized for expression in P.moriformis, was utilized as a selectable marker for transformation. Thesequence of the transforming DNA is provided immediately below. Relevantrestriction sites are indicated in lowercase, bold, and are from 5′-3′BspQI, KpnI, AscI, MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, AscI, ClaI,BglII, AflII, HinDIII, SacI, SpeI, and XhoI, respectively. BspQI sitesdelimit the 5′ and 3′ ends of the transforming DNA. Underlined sequencesat the 5′ and 3′ flanks of the construct represent genomic DNA from P.moriformis that enable targeted integration of the transforming DNA viahomologous recombination at the FATA-1 locus. Proceeding in the 5′ to 3′direction, the C. reinhardtii TUB2 promoter driving the expression ofthe S. cerevisiae SUC2 gene (encoding sucrose hydrolyzing activity,thereby permitting the strain to grow on sucrose) is indicated bylowercase, boxed text. The initiator ATG and terminator TGA for SUC2 areindicated by uppercase italics, while the coding region is indicatedwith lowercase italics. The 3′ UTR of the C. vulgaris nitrate reductase(NR) gene is indicated by small capitals, followed by a spacer regionindicated by lowercase text. The P. moriformis AMT3 promoter, indicatedby lowercase boxed text, drives expression of the P. moriformis KASIIgene. The region encoding the plastid targeting peptide from Chlorellaprotothecoides SAD1 is indicated by uppercase italics. The sequence thatencodes the mature P. moriformis KASII polypeptide is indicated withbold, underlined, uppercase italics, while a 3×FLAG epitope encodingsequence is in bold italics. A second C. vulgaris NR 3′ UTR is indicatedby small capitals. A second C. reinhardtii TUB2 promoter sequence,indicated by lowercase boxed text, drives expression of the P.moriformis FAD2 hairpin A sequence. The sense and antisense strands areindicated with uppercase, bold italics, and are separated by the FAD2intron and the first 10 bases of the FAD2 second exon (uppercaseitalics). A third C. vulgaris NR 3′ UTR is indicated by small capitals,followed by a second spacer region that is indicated by lowercase text.

Nucleotide sequence of the the transforming DNA from pSZ2419: gctcttcggagtcactgtgccactgagttcgactggtagctgaatggagtcgctgctccactaaacgaattgtcagcaccgccagccggccgaggacccgagtcatagcgagggtagtagcgcgccatggcaccgaccagcctgcttgccagtactggcgtctcttccgcttctctgtggtcctctgcgcgctccagcgcgtgcgcttttccggtggatcatgcggtccgtggcgcaccgcagcggccgctgcccatgcagcgccgctgcttccgaacagtggcggtcagggccgcacccgcggtagccgtccgtccggaacccgcccaagagttttgggagcagcttgagccctgcaagatggcggaggacaagcgcatcttcctggaggagcaccggtgcgtggaggtccggggctgaccggccgtcgcattcaacgtaatcaatcgcatgatgatcagaggacacgaagtcttggtggcggtggccagaaacactgtccattgcaagggcatagggatgcgttccttcacctctcatttctcatttctgaatccctccctgctcactctttctcctcctccttcccgt

cgccATGctgctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtgcgcgaggtcaagTGAcaa ttgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttg

CAATGCCCGCTGCGGCGACCTGCGTCGCTCGGCGGGCTCCGGGCCCCGGCGCCCAGCGAGGCCC

AGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGG

TGCCCGTATTGAGGTCCTGGTGGCGCGCATGGAGGAGAAGGCGCCTGTCCCGCTGACCCCCCCG

TGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagctgtattgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaagacagggtggttggctggatggggaaacgctggtcgcgggattcgatcctgctgcttatatcctccctggaagcacacccacgactctgaagaagaaaacgtgcacacacacaacccaaccggccgaatatttgcttccttatcccgggtccaagagagactgcgatgcccccctcaatcagcatcctcctccctgccgcttcaatcttccctgcttgcctgcgcccgcggtgcgccgtctgcccgcccagtcagtcactcctgcacaggccccttgtgcgcagtgctcctgtaccctttaccgctccttccattctgcgaggccccctattgaatgtattcgttgcctgtgtggccaagcgggctgctgggcgcgccgccgtcgggcagtgctcggcgactttggcggaagccgattgttcttcgtaagccacgcgcttgctgctttgggaagagaagggggggggtactgaatggatgaggaggagaaggaggggtattggtattatctgagttgggt gaagagc

Identification and Analysis of FATA-1 Knockout, KASII Over-Expressionand FAD2 RNAi Strains:

Construct D1358, derived from pSZ2419, was transformed into S1920 asdescribed previously. Primary transformants were clonally purified andgrown under standard lipid production conditions at pH 5. The resultingfatty acid profiles from representative clones arising fromtransformation of S1920 with D1358 are summarized in Table 45, below.The P. moriformis AMT3 promoter is repressed at pH 5 so the observedphenotypes did not reflect over-expression of P. moriformis KASII.Nevertheless, we observed that multiple strains had substantiallyreduced levels of C16:0 and 10-15% increases in C18:1, suggesting thatthe construct had disrupted the FATA-1 target gene, increasing theamount of palmitoyl-ACP available for extension by endogenous KASII. Oneline, D1358-13, was selected for further analysis. D1358-13 accumulated˜17% C16:0, ˜75% C18:1 and less than 2% C18:2, indicating that we hadsuccessfully integrated at FATA-1 and down-regulated activity of theFAD2 Δ¹²-desaturase in this strain.

TABLE 45 Fatty acid profiles of D1358 [pSZ2419] primary transformants,compared to the wild-typ parental strain, S1920. D1358- D1358- D1358-D1358- D1358- D1358- D1358- D1358- D1358- D1358- Strain S1920 13 19 11 930 28 6 8 10 3 Fatty C12:0 0.05 0.08 0.06 0.08 0.06 0.07 0.07 0.09 0.070.08 0.10 Acid C14:0 1.32 0.79 0.83 0.85 0.87 0.84 0.91 0.86 0.89 0.920.60 Area C16:0 26.66 17.43 18.84 20.03 16.27 18.4 19.1 18.18 15.6 16.4211.24 % C16:1 0.84 0.74 0.79 0.97 0.60 0.77 1.17 0.75 0.56 0.61 0.57C18:0 3.10 2.87 2.97 2.36 3.20 2.67 2.10 2.82 3.22 3.19 2.30 C18:1 59.0774.78 68.54 68.78 71.48 69.55 69.02 68.93 70.44 69.64 75.27 C18:2 7.391.97 5.47 5.61 6.22 6.31 6.42 6.8 7.68 7.78 8.51 C18:3α 0.55 0.23 0.590.51 0.26 0.39 0.46 0.38 0.24 0.27 0.24 C20:0 0.24 0.22 0.20 0.13 0.320.20 0.03 0.20 0.33 0.31 0.22 C20:1 0.11 0.40 0.29 0.37 0.23 0.33 0.330.39 0.36 0.27 0.40 C22:0 0.11 0.09 0.08 0.07 0.09 0.08 0.08 0.08 0.090.11 0.11 sum C18 70.11 79.85 78.57 77.26 81.16 78.92 78.00 78.93 81.5880.88 86.32 saturates 31.48 21.48 22.98 23.52 20.81 22.26 22.29 22.2320.20 21.03 14.57 unsaturates 67.96 78.12 76.68 76.24 78.79 77.35 77.477.25 79.28 78.57 84.99

In Table 45, Oleate (C18:1) levels greater than the wild-type level arehighlighted with bold text. Palmitate (C16:0) levels less than thewild-type are highlighted with bold text. Levels of linoleate (C18:2)reduced by 1% or more compared to the S1920 parent are highlighted withbold text.

The fatty acid profiles of strains derived from transformant D1358-13were determined to be stable after more than 60 generations of growth inthe absence of selection (growth on sucrose). The performance ofselected strains in shake flask assays was then evaluated, and the fattyacid profiles and lipid titers are presented in Table 46, below. Flaskexperiments were performed at pH 7, enabling activation of the PmAMT3promoter driving expression of the KASII transgene. The combination ofKASII over-expression and FATA-1 knockout leads to further reductions inpalmitate levels and enhanced oleate accumulation compared to thephenotypes observed at pH 5 (Table 45). With more than 82% C18:1, lessthan 11% C16:0, less than 2% C18:2 and ˜83% of the wild-type lipidtiter, S5003 was determined to be the most appropriate strain from thisset to serve as a host strain for subsequent modifications to elevatestearate levels. DNA blot analysis showed that S5003 has a simpleinsertion of construct D1358 [pSZ2419] at the FATA-1 locus.

TABLE 46 Fatty acid profiles and lipid titers of FATA-1 knockout, KASIIover- expressing, FAD2 RNAi lines from D1358-13 primary transformants,compared to the wild-type parental strain, S1920. Primary T389, D1358-13Strain S1920 S5003 S5004 S5005 S5006 S5007 S5008 S5009 S5010 S5011 S5012S5013 S5101 S5102 Fatty C12:0 0.05 0.08 0.09 0.11 0.19 0.11 0.14 0.100.12 0.08 0.11 0.09 0.20 0.20 Acid C14:0 1.34 0.96 0.98 1.03 1.04 0.961.02 0.98 1.03 0.98 1.01 1.00 1.03 1.02 Area C16:0 29.69 10.72 10.478.90 6.99 9.53 9.27 10.13 8.99 10.76 9.58 10.00 6.64 6.38 % C16:1 0.880.42 0.39 0.31 0.29 0.39 0.37 0.41 0.32 0.40 0.35 0.35 0.27 0.27 C18:02.78 2.92 3.00 3.16 2.71 2.88 2.85 2.91 3.21 3.03 3.10 3.20 2.77 2.71C18:1 58.45 82.08 82.24 83.66 85.49 83.28 83.38 82.57 83.51 82.12 83.1082.63 85.88 86.13 C18:2 5.83 1.89 1.88 1.80 2.01 1.83 1.89 1.89 1.771.73 1.75 1.76 1.94 1.96 C18:3α 0.42 0.23 0.23 0.25 0.35 0.27 0.29 0.270.25 0.22 0.24 0.23 0.34 0.36 C20:0 0.17 0.15 0.16 0.17 0.15 0.15 0.160.16 0.17 0.14 0.16 0.16 0.15 0.15 C20:1 0.05 0.23 0.24 0.27 0.36 0.280.29 0.26 0.27 0.21 0.25 0.24 0.38 0.39 sum C18 67.48 87.12 87.35 88.8790.56 88.26 88.41 87.64 88.74 87.10 88.19 87.82 90.93 91.16 saturates34.03 14.83 14.70 13.37 11.08 13.63 13.44 14.28 13.52 14.99 13.96 14.4510.79 10.46 unsaturates 65.63 84.85 84.98 86.29 88.50 86.05 86.22 85.4086.12 84.68 85.69 85.21 88.81 89.11 lipid titer (% 100.0 82.8 81.1 72.854.4 68.3 63.7 70.6 72.2 106.9 76.5 77.5 56.7 54.6 parent)

In Table 46, Stearate (C18:1) levels greater than the wild-type levelare highlighted with bold text. Palmitate (C16:0) levels lower than thewild-type are highlighted with bold text. Linoleate (C18:2) levels thatare lower than the wild-type are indicated with bold text.

Constructs Used for SAD2 Knockout/RNAi in S5003:

Two DNA constructs, pSZ2283 and pSZ2697, were made to simultaneouslydisrupt the SAD2-1 allele and express a SAD2 hairpin construct in S5003.In each construct, the neoR gene from transposon Tn5, conferringresistance to aminoglycoside antibiotics, was used as a selectablemarker for transformation. The sequence of the transforming DNA derivedfrom pSZ2283 is provided immediately below. Relevant restriction sitesare indicated in lowercase, bold, and are from 5′-3′ BspQI, KpnI, XbaI,MfeI, BamHI, AvrII, EcoRV, EcoRI, SpeI, BamHI, HinDIII, and SacI,respectively. BspQI sites delimit the 5′ and 3′ ends of the transformingDNA. Underlined sequences at the 5′ and 3′ flanks of the constructrepresent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the Chlamydomonasreinhardtii TUB2 promoter driving the expression of neoR (encodingaminoglycoside phosphotransferase activity, thereby permitting thestrain to grow on G418) is indicated by lowercase, boxed text. Theinitiator ATG and terminator TGA for neoR are indicated by uppercaseitalics, while the coding region is indicated with lowercase italics.The 3′ UTR of the C. vulgaris NR gene is indicated by small capitals,followed by a spacer region indicated by lowercase text. A second C.reinhardtii TUB2 promoter sequence, indicated by lowercase boxed text,drives expression of the SAD2 hairpin C sequence. The sense andantisense strands are indicated with uppercase, bold italics, and areseparated by the P. moriformis FAD2 intron and the first 10 bases of theFAD2 second exon (uppercase italics). A second C. vulgaris NR 3′ UTR isindicated by small capitals.

Nucleotide sequence of the the transforming DNA from pSZ2283: gctcttcgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgca

ccggctcccccgccgcctgggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggag

GATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAaagctgga gctccagccacggcaacaccgcgcgccttgcggccgagcacggcgacaagaacctgagcaagatctgcgggctgatcgccagcgacgagggccggcacgagatcgcctacacgcgcatcgtggacgagttcttccgcctcgaccccgagggcgccgtcgccgcctacgccaacatgatgcgcaagcagatcaccatgcccgcgcacctcatggacgacatgggccacggcgaggccaacccgggccgcaacctcttcgccgacttctccgcggtcgccgagaagatcgacgtctacgacgccgaggactactgccgcatcctggagcacctcaacgcgcgctggaaggtggacgagcgccaggtcagcggccaggccgccgcggaccaggagtacgtcctgggcctgccccagcgcttccggaaactcgccgagaagaccgccgccaagcgcaagcgcgtcgcgcgcaggcccgtcgccttctcctggatctccgggcgcgagatcatggtctagggagcgacgagtgtgcgtgcggggctggcgggagtgggacgccctcctcgctcctctctgttctgaacggaacaatcggccaccccgcgctacgcgccacgcatcgagcaacgaagaaaaccccccgatgataggttgcggtggctgccgggatatagatccggccgcacatcaaagggcccctccgccagagaagaagctcctttcccagcagactcctgaagagc

The sequence of the transforming DNA derived from pSZ2697 is providedimmediately below. Relevant restriction sites are indicated inlowercase, bold, and are from 5′-3′ NsiI, SpeI, BamHI, HinDIII, SacII,EcoRV, KpnI, XbaI, MfeI, BamHI, AvrII, EcoRV, EcoRI and XbaI,respectively. Underlined sequences at the 5′ and 3′ flanks of theconstruct represent genomic DNA from P. moriformis that enable targetedintegration of the transforming DNA via homologous recombination at theSAD2-1 locus. Proceeding in the 5′ to 3′ direction, the SAD2 hairpin Csense and antisense strands are indicated with uppercase, bold italics,and are separated by the P. moriformis FAD2 intron and the first 10bases of the FAD2 second exon (uppercase italics). The 3′ UTR of the C.vulgaris NR gene is indicated by small capitals. The Chlorellasorokiniana Glutamate Dehydrogenase (GDH) promoter, driving theexpression of neoR (encoding aminoglycoside phosphotransferase activity,thereby permitting the strain to grow on G418) is indicated bylowercase, boxed text. The initiator ATG and terminator TGA for neoR areindicated by uppercase italics, while the coding region is indicatedwith lowercase italics. A second C. vulgaris NR 3′ UTR is indicated bysmall capitals, followed by a spacer region indicated by lowercase text.

Nucleotide sequence of the the transforming DNA from pSZ2697: atgcatgccggtcaccacccgcatgctcgtactacagcgcacgcaccgcttcgtgatccaccgggtgaacgtagtcctcgacggaaacatctggttcgggcctcctgcttgcactcccgcccatgccgacaacctttctgctgttaccacgacccacaatgcaacgcgacacgaccgtgtgggactgatcggttcactgcacctgcatgcaattgtcacaagcgcttactccaattgtattcgtttgttttctgggagcagttgctcgaccgcccgcgtcccgcaggcagcgatgacgtgtgcgtggcctgggtgtttcgtcgaaaggccagcaaccctaaatcgcaggcgatccggagattgggatctgatccgagtttggaccagatccgccccgatgcggcacgggaactgcatcgactcggcgcggaacccagattcgtaaatgccagattggtgtccgatacctggatttgccatcagcgaaacaagacttcagcagcgagcgtatttggcgggcgtgctaccagggttgcatacattgcccatttctgtctggaccgctttactggcgcagagggtgagttgatggggttggcaggcatcgaaacgcgcgtgcatggtgtgcgtgtctgttttcggctgcacgaattcaatagtcggatgggcgacggtagaattgggtgtggcgctcgcgtgcatgcctcgccccgtcgggtgtcatgaccgggactggaatcccccctcgcgaccatcttgct

GGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAAAGCT

gggtggagcgcctgttcggctacgactgggcccagcagaccatcggctgctccgacgccgccgtgttccgcctgtccgcccagggccgccccgtgctgttcgtgaagaccgacctgtccggcgccctgaacgagctgcaggacgaggccgcccgcctgtcctggctggccaccaccggcgtgccctgcgccgccgtgctggacgtggtgaccgaggccggccgcgactggctgctgctgggcgaggtgcccggccaggacctgctgtcctcccacctggcccccgccgagaaggtgtccatcatggccgacgccatgcgccgcctgcacaccctggaccccgccacctgccccttcgaccaccaggccaagcaccgcatcgagcgcgcccgcacccgcatggaggccggcctggtggaccaggacgacctggacgaggagcaccagggcctggcccccgccgagctgttcgcccgcctgaaggcccgcatgcccgacggcgaggacctggtggtgacccacggcgacgcctgcctgcccaacatcatggtggagaacggccgcttctccggcttcatcgactgcggccgcctgggcgtggccgaccgctaccaggacatcgccctggccacccgcgacatcgccgaggagctgggcggcgagtgggccgaccgcttcctggtgctgtacggcatcgccgcccccgactcccagcgcatcgccttctaccgcctgctggacgagttcttcTGAcaattgGCAGCAGCAGCTCGGATAGTATCGACACACTCTGGACGCTGGTCGTGTGATGGACTGTTGCCGCCACACTTGCTGCCTTGACCTGTGAATATCCCTGCCGCTTTTATCAAACAGCCTCAGTGTGTTTGATCTTGTGTGTACGCGCTTTTGCGAGTTGCTAGCTGCTTGTGCTATTTGCGAATACCACCCCCAGCATCCCCTTCCCTCGTTTCATATCGCTTGCATCCCAACCGCAACTTATCTACGCTGTCCTGCTATCCCTCAGCGCTGCTCCTGCTCCTGCTCACTGCCCCTCGCACAGCCTTGGTTTGGGCTCCGCCTGTATTCTCCTGGTACTGCAACCTGTAAACCAGCACTGCAATGCTGATGCACGGGAAGTAGTGGGATGGGAACACAAATGGAggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccacgttggcgaggtggcaggtgacaatgatcggtggagctgatggtcgaaacgttcacagcctagggatatcgaattccgggtcgccgcgctgcctcgcgtcccctggtggtgcgcgcggtcgccagcgaggccccgctgggcgttccgccctcggtgcagcgcccctcccccgtggtctactccaagctggacaagcagcaccgcctgacgcccgagcgcctggagctggtgcagagcatggggcagtttgcggaggagagggtgctgcccgtgctgcaccccgtggacaagctgtggcagccgcaggactttttgcccgaccccgagtcgcccgacttcgaggatcaggtggcggagctgcgcgcgcgcgccaaggacctgcccgacgagtactttgtggtgctggtgggggacatgatcacggaggaggcgctgccgacctacatggccatgctcaacacgctggacggcgtgcgcgacgacacgggcgcggccgaccacccgtgggcgcgctggacgcggcagtgggtggccgaggagaaccggcacggcgacctgctgaacaagtactgctggctgacggggcgcgtcaacatgcgggccgtggaggtgaccatcaacaacctgatcaagagcggcatgaacccgcagacggacaacaacccttatttggggttcgtctacacctccttccaggagcgcgccaccaagtatct aga

Identification and Analysis of SAD2 Knockout/Knockdown Strains in theS5003 Background:

Constructs D1639, derived from pSZ2697, and D1682, derived from pSZ2283,were transformed into S5003 as described previously. Primarytransformants were clonally purified and grown under standard lipidproduction conditions at pH 7. The resulting fatty acid profiles fromrepresentative clones arising from transformation are summarized inTable 47, below. D1639 transformants accumulated up to 56% C18:0, andD1682 transformants accumulated a maximum of about 35% C18:0. Most ofthe increases in stearate came at the expense of C18:1, indicating thatSAD activity was significantly reduced by the SAD2 knockout/RNAiconstructs in these strains. C16:0 levels varied from 6% to 14%; C18:2ranged from 2-5%. Most strains maintained the low C16:0 and C18:2phenotypes of the S5003 parent. These fatty acid profiles demonstratethat down-regulating SAD2 expression using knockout/RNAi constructs, ina background with disrupted FATA-1, KASII over-expression and FAD2 RNAi,produces strains with high C18:0, low C16:0 and low C18:2 phenotypes.These strains will be useful for production of high stability, highstearate, high oleic oils, and oils which have high SOS content.

TABLE 47 Fatty acid profiles of D1639 [pSZ2697] and D1682 [pSZ2283]primary transformants, compared to the wild-type strain, S1920, and theS5003 parental base strain. Strain S1920 S5003 D1682-4 D1682-17 D1682-7D1682-6 D1639-2 D1639-5 D1639-10 D1639-19 Fatty C12:0 0.04 0.11 0.140.10 0.32 0.31 0.00 0.19 0.17 0.00 Acid C14:0 1.29 0.98 1.03 0.94 1.111.15 1.64 1.39 1.61 1.02 Area C16:0 27.50 7.75 8.68 10.41 5.70 5.96 7.549.90 14.39 12.02 % C16:1 0.71 0.30 0.06 0.07 0.07 0.10 0.00 0.00 0.000.00 C18:0 3.28 3.60 35.46 29.92 24.66 22.30 55.96 53.38 43.46 37.30C18:1 57.80 84.14 48.39 52.49 61.04 63.60 23.70 26.79 32.93 42.81 C18:27.90 2.09 2.37 2.36 3.03 2.88 5.09 3.50 3.22 2.79 C18:3α 0.57 0.32 0.500.65 0.66 0.58 1.59 0.98 1.01 0.85 C20:0 0.28 0.23 2.07 1.87 1.75 1.513.04 2.73 2.29 2.22 C20:1 0.18 0.35 0.54 0.49 0.78 0.83 0.37 0.33 0.300.40 C22:0 0.06 0.02 0.27 0.27 0.23 0.20 0.43 0.36 0.29 0.29 C24:0 0.090.02 0.33 0.26 0.34 0.26 0.64 0.45 0.32 0.31 sum C18 69.55 90.14 86.7285.42 89.39 89.36 86.34 84.65 80.62 83.75 saturates 32.54 12.70 47.9843.77 34.11 31.69 69.25 68.40 62.53 53.16 unsaturates 67.16 87.21 51.8656.06 65.58 67.99 30.75 31.60 37.46 46.85

In Table 47, Stearate (C18:0) levels greater than the wild-type levelare highlighted with bold text. Oleate (C18:1) levels that are higherthan in the wild-type are indicated with bold text. Palmitate (C16:0)levels less than the wild-type level are highlighted with bold. Reducedlevels of linoleate (C18:2) compared to the wild-type are highlightedwith bold text.

Stable lines were isolated from a number of D1639 and D1682transformants. Shake flask assays were carried out to evaluate theperformance of four lines derived from D1639-5. Fatty acid profiles andrelative lipid titers from the biomass are shown in Table 48, below.

TABLE 48 Shake flask assays of strains derived from D1639-5, expressingSAD2hpC, driven by the CrTUB2 promoter, targeted to the SAD2-1 locus.Primary T530; D1639-5 Strain S1920 S5003 S5774 S5775 S5776 S5777 FattyC10:0 0.01 0.00 0.07 0.08 0.05 0.04 Acid C12:0 0.02 0.11 0.19 0.22 0.250.23 Area C14:0 1.52 1.10 1.35 1.32 1.30 1.43 % C16:0 31.61 9.59 9.288.44 7.74 9.46 C16:1 1.04 0.34 0.03 0.02 0.01 0.01 C17:0 0.10 0.11 0.100.10 0.10 0.09 C18:0 2.98 4.36 53.01 53.52 55.32 52.09 C18:1 54.81 80.8427.26 27.52 27.42 28.06 C18:2 6.88 2.42 3.55 3.52 2.38 3.45 C18:3α 0.530.33 0.97 1.03 0.82 1.06 C20:0 0.26 0.31 2.88 2.94 3.15 2.72 C20:1 0.030.06 0.36 0.37 0.39 0.35 C22:0 0.07 0.08 0.53 0.54 0.53 0.60 C24:0 65.1987.95 84.79 85.58 85.94 84.66 sum C18 36.59 15.70 67.76 67.52 68.8266.99 saturates 63.30 84.26 32.19 32.46 31.02 32.95 unsaturates 100.070.3 34.8 33.7 31.4 35.3 % wild-type lipid titer

In Table 48, S5003 is the parent strain; S1920 is the wild-type basestrain. Stearate (C18:0) levels higher than in the wild-type strain areindicated with bold. Bold text indicates the increased level of oleate(C18:1) in S5003 compared to the wild-type. Palmitate (C16:0) levelsthat are less than in the wild-type are highlighted bold. Linoleate(C18:2) levels that are less than in the wild-type are indicated withbold.

Lab scale oils were prepared from biomass collected from the S5774,S5775 and S5776 shake flasks. The TAG compositions of these oils weredetermined by LC/MS, and are shown in FIG. 21. SOS accumulation rangedfrom 42-47% in these strains. POS was the next most abundant TAG, at16-17%. Linoleate-containing TAGs were reduced by more than 50% comparedto the S5665 and S5675 oils, described above. S5774-S5776 oils contained12-13% trisaturated TAGs (S-S-S), similar to the amounts thataccumulated in the S5665 and S5775 oils. Modulation of SAD activityduring oil production to prevent overproduction of saturated fatty acidsmay help to reduce accumulation of trisaturates.

Example 49 Properties of Methyl Oleate from High Oleic Microalgal Oils

Esterified oils high in methyl oleate are useful in a variety ofapplications such as cleaning and lubrication of machinery. For some ofthese applications, high thermal stability is desired. Thermal stabilitytesting was performed on methylated oil prepared from high-oleic andhigh-stability-high oleic triglyceride oils prepared fromheterotrophically grown oleaginous microalgae as described above. Theoils were bleached and deodorized prior to methylation. Commericallyavailable soya methyl ester was used as a control.

High Oleic (HO) oil was prepared from a high oil-yielding strain ofPrototheca moriformis transformed with a plasmid that can be describedas FatA1_Btub:inv:nr::amt03-CwTE2:nr_FatA1. This plasmid was designed tohomologously recombine in the FATA1 chromosomal site, thus ablating aFATA acyl-ACP thioesterase choromosomal allele, while expressing anexogenous acyl-ACP thioesterase from Cuphea. wrightii (CwTE2, SEQ ID NO:11) under control of the pH-regulatable amt3 promoter. The CwTE2 genecan be downregulated by cultivation at pH 5 during oil production tofurther elevate oleate production. Sucrose invertase was also expressedas a selection marker and to allow for cultivation of the strain onsucrose as a sole carbon source. The 3′ UTR sequences are from theChlorella vulgaris nitrate reductase gene. The resulting HO strain isdenoted Stain Q. The fatty acid profile of the oil produced by Strain Qis listed below in Table 49.

TABLE 49 Fatty acid profile of high oleic oil from Strain Q. Fatty AcidArea % C10 0.01 C12:0 0.03 C14:0 0.43 C15:0 0.03 C16:0 7.27 C16:1 iso0.81 C16:1 0.689 C17:0 0.06 C18:0 1.198 C18:1 80.15 C18:1 iso 0.08 C18:28.38 C18:3 ALPHA 0.25 C20:0 0.02 C20:1 0.38 C22:0 0.04 C24:0 0.03

A high-stability-high-oleic oil (HSAO) was also prepared from a highoil-yielding strain of Prototheca moriformis transformed with a plasmidthat can be described as FADc5′_Btub:inv:nr::btub-CpSAD_CtOTE:nr_FADc3′.The resulting strain (Strain R) expresses sucrose invertase as aselection marker and to allow for cultivation on sucrose as a solecarbon source. In addition, a FAD allele (encoding fatty acid desaturaseresponsible for the conversion of oleate to linoleate) is disrupted andan oleate-specific acy-ACP thioesterase (Carthamus tinctorius OTE, seeexample 5) fused to the transit peptide from the SAD gene of Chlorellaprotothecoides is expressed under control of the beta tubulin promoter.The 3′ UTR sequences are from the Chlorella vulgaris nitrate reductasegene. The fatty acid profile of the oil produced by Strain R afterheterotrophic cultivation is listed below in Table 50. The fatty acidprofile has greater than 85% oleate yet almost none of the majorpolyunsaturates, linoeic and linolenic acids.

TABLE 50 Fatty acid profile of high oleic oil from Strain R. Fatty AcidArea % C10 0.02 C12:0 0.07 C14:0 0.09 C15:0 0.05 C16:0 7.28 C16:1 0.70C17:0 0.08 C18:0 2.15 C18:1 86.32 C20:0 0.30 C20:1 0.46 C22:0 0.08 C23:00.01 C24:0 0.06

The HO and HSAO oils were methylated by known biodiesel productiontechniques to make methyl-HO and methyl-HSAO esters. These methyl esterswhere then subjection to thermal testing according to the followingprocedure:

-   -   1. Prepare equipment as shown in FIG. 1.    -   2. Add 1 litre of water to test vessel and bring to an active        boil on the hotplate.    -   3. To each test product add 50 ppm Cobalt (0.083 g of 6% Cobalt        Napthenate in 100.0 gram sample) and mix thoroughly.    -   4. Weigh out, in a watch glass, 7.0 g of 100% cotton gauze, (#50        Cheese Cloth).    -   5. Evenly distribute 14.0 g of test product, as prepared in step        3, onto the gauze.    -   6. Place thermocouple (thermometer) through the center of #15        stopper. Wrap cotton around the thermocouple.    -   7. Place wrapped cotton into 24 mesh wire frame cylinder so that        it occupies the upper 4½ inches.    -   8. Position cylinder with wrapped gauze into the 1 L tall form        beaker. Secure the beaker in the boiling water and begin        recording the temperature increase with time.    -   9. Continue monitoring the temperature for 2 hours or until a 10        degree temperature drop in observed.    -   10. Plot temperature vs time on a graph.    -   11. Any sample which shows a temperature exceeding 100        degrees C. in 1 hour or 200 degrees C. in 2 hours should be        regarded as a dangerous oxidation risk or one that is likely to        spontaneously combust.

Results: The HO and HSAO methyl ester did not exhibit auto-oxidation asevidenced by a temperature rise. The control soya methyl ester sampledid exhibit the potential for auto-oxidation. The time-temperatureprofiles are shown in FIG. 18.

In addition, methylated fatty acid from oil produced by Strain Q wasfound to have the following characteristics:

Flash Point (ASTM D93) of 182° C.

Non-VOC

Kauri Butanol value (ASTM D1133) of 53.5

Viscosity at 40° C. (ASTM D445) of 4.57 mm2/s

Acid Number (ASTM D664) of 0.17 mg KOH/g

Boiling range distribution (ASTM D2887) 325-362° C.

Example 50 Further Properties of High Oleic (HO) andHigh-Stability-High-Oleic (HSAO) Microalgal Oils

The high oleic oil and the high-stability high-oleic algal oils can havethe properties shown in FIG. 19 or these values±20% for the measuredparameters.

In one experiment, HSAO microalgal oil showed 512 hour stabilitymeasured by OSI at 110° C. (estimated using 130° C. data) withantioxidants of 0.5% phenyl-alpha-naphthylamine (PANA) and 500 ppmascorbyl palmitate (AP).

Example 51 Production of Low Saturate Oil by Conversion of Palmitic toPalmitoleate

As described in the examples above, genetic manipulation of microalgaecan decrease saturated fat levels, especially by increasing theproduction of oleic acid. However, in some cases, the acyl-ACPthioesterases expressed in the oleaginous cell liberate more thandesirable amounts of palmitate. Here, we describe methods for convertingpalmitate (16:0) to palmitoleate (16:1) by overexpressing apalmitoyl-ACP desaturase (PAD) gene. The PAD gene can be obtained fromnatural sources such as Macfadyena unguis (Cat's claw), Macadamiaintegrifolia (Macadamia nut), Hippophae rhamnoides (sea buckthorn), orby creating a PAD via mutation of a stearoyl-ACP desaturase to have 16:1activity. The Macfadyena unguis desaturase is denoted (MuPAD).

A high-oil-producing strain of Prototheca moriformis (Strain Z) isbiolistically transformed with plasmid DNA constructs with a PAD gene.For example, one of the high oleic strains described in the Examples 6,36, or 49 can further comprise an exogenous PAD gene. The constructscomprises sucrose invertase as a selectable marker and either the MuPADor a SAD gene (e.g., Olea europaea stearoyl-ACP desaturase, GenBankAccession No. AAB67840.1) having the L118W mutation to shiftsubstrate-specificity toward palmitate. See Cahoon, et al., PlantPhysoil (1998) 117:593-598. Both the amt3 and beta tubulin (Btub)promoters are used. In addition, the native transit peptide of a plantPAD gene can be swapped with one known to be effective in microalgae(e.g., the transit peptide from the Chlorella vularis SAD gene).

The PAD gene can be expressed in a variety of strains including thosewith a FATA knockout or knockdown and/or a KASII knockin to producehigh-oleic oil. Optionally, these strains can also producehigh-stability (low polyunsaturate) oil by virtue of a FAD (delta 12fatty acid desaturase) knockout, knockdown, or by placing FAD expressionunder control of a regulatable promoter and producing oil underconditions that downregulate FAD. In addition, useful base strains forthe introduction of PAD gene activities might also include strainspossessing KASII knockouts, and FATA Knockins, whereby levels of C16:0palmitate are elevated.

As a result, lower levels of palmitic acid are found in the fatty acidprofile of the microalgal oil as this is converted into cis-palmitoleicand cis-vaccenic acids. In some cases the total area percent ofsaturated fatty acids is less than equal to 3.5%, 3% or 2.5%.

Constructs for over expression of Macfadyena unguis C16:0 desaturase(MuPAD) follow:

1) pSZ3142:6S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ31426S::CrTUB2:ScSUC2:CvNR::PmAMT3:CpSADtp:MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of Prototheca moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the MuPAD are indicated byuppercase, bold italics, while the remainder of the coding region isindicated by bold italics. The Chlorella protothecoides S106stearoyl-ACP desaturase transit peptide is located between initiator ATGand the Asc I site. The C. vulgaris nitrate reductase 3′ UTR is againindicated by lowercase underlined text followed by the 6S genomic regionindicated by bold, lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3142: gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcct

tcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgt

gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccac

gtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

2) pSZ3145:6S::CrTUB2:ScSUC2:CvNR::PmAMT3:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ31456S::CrTUB2:ScSUC2:CvNR::PmAMT3:MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Cla I, Sac I, BspQ I, respectively. BspQIsites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by an endogenous amt03promoter of Prototheca moriformis, indicated by boxed italics text. TheInitiator ATG and terminator TGA codons of the MuPAD are indicated byuppercase, bold italics, while the remainder of the coding region isindicated by bold italics. The C. vulgaris nitrate reductase 3′ UTR isagain indicated by lowercase underlined text followed by the 6S genomicregion indicated by bold, lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3145: gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcct

tcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgt

gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccac

tgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagctt gaagagc

3) pSZ3137:6S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S

Relevant restriction sites in the construct pSZ3137

6S::CrTUB2:ScSUC2:CvNR::CrTUB2:CpSADtp:MuPAD:CvNR::6S are indicated inlowercase, bold and underlining and are 5′-3′ BspQ 1, Kpn I, Xba I, MfeI, BamH I, EcoR I, Spe I, Asc I, Cla I, Sac I, BspQ I, respectively.BspQI sites delimit the 5′ and 3′ ends of the transforming DNA. Bold,lowercase sequences represent genomic DNA from that permit targetedintegration at 6s locus via homologous recombination. Proceeding in the5′ to 3′ direction, the C. reinhardtii β-tubulin promoter driving theexpression of the yeast sucrose invertase gene (conferring the abilityof Strain Z to metabolize sucrose) is indicated by boxed text. Theinitiator ATG and terminator TGA for invertase are indicated byuppercase, bold italics while the coding region is indicated inlowercase italics. The Chlorella vulgaris nitrate reductase 3′ UTR isindicated by lowercase underlined text followed by C. reinhardtiiβ-tubulin promoter, indicated by boxed italics text. The Initiator ATGand terminator TGA codons of the MuPAD are indicated by uppercase, bolditalics, while the remainder of the coding region is indicated by bolditalics. The Chlorella protothecoides S106 stearoyl-ACP desaturasetransit peptide is located between initiator ATG and the Asc I site. TheC. vulgaris nitrate reductase 3′ UTR is again indicated by lowercaseunderlined text followed by the 6S genomic region indicated by bold,lowercase text.

Nucleotide sequence of transforming DNA contained in pSZ3137: gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgctgatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggaggactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaactggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacgagccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgccgcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagtcggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccaccccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcct

tcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgcacttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagtacaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagcccatcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacgacaccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctggacggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtacgagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctggaagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggaccccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttcaacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaacaccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctcctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagccgatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacctgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacctctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctccttcttcctggaccgcgggaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaacgacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacacctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgt

gctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaggatcccgcgtctcgaacagagcgcgcagaggaacgctgaaggtctcgcctctgtcgcacctcagcgcggcatacaccacaataaccacctgacgaatgcgcttggttcttcgtccattagcgaagcgtccggttcacacacgtgccac

acacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaaagcttaattaa gagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgataacctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgttgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaatcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgccaggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctcgaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatggggctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacctgctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgcttctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc

The described embodiments of the invention are intended to be merelyexemplary and numerous variations and modifications will be apparent tothose skilled in the art. All such variations and modifications areintended to be within the scope of the present invention. For example,the various triglyceride oils can be tailored in for a mixture ofmidchain and long chain fatty acids in order to adjust parameters suchas polarity, solvency, and foam-height of the oils or chemicals madefrom the oils.

1. A method for producing an oil comprising: cultivating a recombinantoleaginous cell comprising recombinant nucleic acids operable to producea palmitoyl-ACP desaturase or variant stearoyl ACP desaturase havingpalmitoyl desaturation activity, wherein the oil produced comprises atleast 1% palmitoleic or vaccenic acid.
 2. The method of claim 1, whereinthe cell further comprises nucleic acids operable to reduce theexpression of a FATA gene encoding an acyl-ACP thioesterase.
 3. Themethod of claim 1, wherein the cell further comprises nucleic acidsoperable to express a KASII enzyme.
 4. The method of claim 1, whereinthe cell further comprises nucleic acids operable to reduce the activityof Δ12 fatty acid desaturase.
 5. An oil produced by the method of claim1.